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Abstract:

The present invention provides novel, stable lipid particles comprising
one or more active agents or therapeutic agents, methods of making the
lipid particles, and methods of delivering and/or administering the lipid
particles. More particularly, the present invention provides stable
nucleic acid-lipid particles (SNALP) comprising a nucleic acid (such as
one or more interfering RNA), methods of making the SNALP, and methods of
delivering and/or administering the SNALP.

Claims:

1-46. (canceled)

47. A nucleic acid-lipid particle comprising: (a) a nucleic acid; (b) a
cationic lipid comprising from 50 mol % to 65 mol % of the total lipid
present in the particle; (c) a non-cationic lipid comprising a mixture of
a phospholipid and cholesterol or a derivative thereof, wherein the
phospholipid comprises from 3 mol % to 15 mol % of the total lipid
present in the particle and the cholesterol or derivative thereof
comprises from 30 mol % to 40 mol % of the total lipid present in the
particle; and (d) a conjugated lipid that inhibits aggregation of
particles comprising from 0.5 mol % to 2 mol % of the total lipid present
in the particle.

Description:

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. application Ser.
No. 12/424,367 filed Apr. 15, 2009 which application claims priority to
U.S. Provisional Application No. 61/045,228, filed Apr. 15, 2008, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] Not applicable.

REFERENCE TO A "SEQUENCE LISTING"

[0004] Not applicable.

BACKGROUND OF THE INVENTION

[0005] RNA interference (RNAi) is an evolutionarily conserved process in
which recognition of double-stranded RNA (dsRNA) ultimately leads to
posttranscriptional suppression of gene expression. This suppression is
mediated by short dsRNA, also called small interfering RNA (siRNA), which
induces specific degradation of mRNA through complementary base pairing.
In several model systems, this natural response has been developed into a
powerful tool for the investigation of gene function (see, e.g., Elbashir
et al., Genes Dev., 15:188-200 (2001); Hammond et al., Nat. Rev. Genet.,
2:110-119 (2001)). More recently, it was discovered that introducing
synthetic 21-nucleotide dsRNA duplexes into mammalian cells could
efficiently silence gene expression.

[0006] Although the precise mechanism is still unclear, RNAi provides a
potential new approach to downregulate or silence the transcription and
translation of a gene of interest. For example, it is desirable to
modulate (e.g., reduce) the expression of certain genes for the treatment
of neoplastic disorders such as cancer. It is also desirable to silence
the expression of genes associated with liver diseases and disorders such
as hepatitis. It is further desirable to reduce the expression of certain
genes for the treatment of atherosclerosis and its manifestations, e.g.,
hypercholesterolemia, myocardial infarction, and thrombosis.

[0010] Other liposomal delivery systems include, for example, the use of
reverse micelles, anionic liposomes, and polymer liposomes. Reverse
micelles are disclosed in U.S. Pat. No. 6,429,200. Anionic liposomes are
disclosed in U.S. Patent Publication No. 20030026831. Polymer liposomes
that incorporate dextrin or glycerol-phosphocholine polymers are
disclosed in U.S. Patent Publication Nos. 20020081736 and 20030082103,
respectively.

[0011] A gene delivery system containing an encapsulated nucleic acid for
systemic delivery should be small (i.e., less than about 100 nm diameter)
and should remain intact in the circulation for an extended period of
time in order to achieve delivery to affected tissues. This requires a
highly stable, serum-resistant nucleic acid-containing particle that does
not interact with cells and other components of the vascular compartment.
The particle should also readily interact with target cells at a disease
site in order to facilitate intracellular delivery of a desired nucleic
acid.

[0012] Recent work has shown that nucleic acids can be encapsulated in
small (e.g., about 70 nm diameter) "stabilized plasmid-lipid particles"
(SPLP) that consist of a single plasmid encapsulated within a bilayer
lipid vesicle (Wheeler et al., Gene Therapy, 6:271 (1999)). These SPLPs
typically contain the "fusogenic" lipid dioleoylphosphatidylethanolamine
(DOPE), low levels of cationic lipid, and are stabilized in aqueous media
by the presence of a poly(ethylene glycol) (PEG) coating. SPLPs have
systemic application as they exhibit extended circulation lifetimes
following intravenous (i.v.) injection, accumulate preferentially at
distal tumor sites due to the enhanced vascular permeability in such
regions, and can mediate transgene expression at these tumor sites. The
levels of transgene expression observed at the tumor site following i.v.
injection of SPLPs containing the luciferase marker gene are superior to
the levels that can be achieved employing plasmid DNA-cationic liposome
complexes (lipoplexes) or naked DNA.

[0013] Thus, there remains a strong need in the art for novel and more
efficient methods and compositions for introducing nucleic acids such as
siRNA into cells. In addition, there is a need in the art for methods of
downregulating the expression of genes of interest to treat or prevent
diseases and disorders such as cancer and atherosclerosis. The present
invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention provides novel, serum-stable lipid particles
comprising one or more active agents or therapeutic agents, methods of
making the lipid particles, and methods of delivering and/or
administering the lipid particles (e.g., for the treatment of a disease
or disorder).

[0015] In preferred embodiments, the active agent or therapeutic agent is
fully encapsulated within the lipid portion of the lipid particle such
that the active agent or therapeutic agent in the lipid particle is
resistant in aqueous solution to enzymatic degradation, e.g., by a
nuclease or protease. In other preferred embodiments, the lipid particles
are substantially non-toxic to mammals such as humans.

[0016] In one aspect, the present invention provides lipid particles
comprising: (a) one or more active agents or therapeutic agents; (b) one
or more cationic lipids comprising from about 50 mol % to about 85 mol %
of the total lipid present in the particle; (c) one or more non-cationic
lipids comprising from about 13 mol % to about 49.5 mol % of the total
lipid present in the particle; and (d) one or more conjugated lipids that
inhibit aggregation of particles comprising from about 0.5 mol % to about
2 mol % of the total lipid present in the particle.

[0017] More particularly, the present invention provides serum-stable
nucleic acid-lipid particles (SNALP) comprising a nucleic acid (e.g., one
or more interfering RNA molecules such as siRNA, aiRNA, and/or miRNA),
methods of making the SNALP, and methods of delivering and/or
administering the SNALP (e.g., for the treatment of a disease or
disorder).

[0018] In certain embodiments, the nucleic acid-lipid particle (e.g.,
SNALP) comprises: (a) a nucleic acid (e.g., an interfering RNA); (b) a
cationic lipid comprising from about 50 mol % to about 85 mol % of the
total lipid present in the particle; (c) a non-cationic lipid comprising
from about 13 mol % to about 49.5 mol % of the total lipid present in the
particle; and (d) a conjugated lipid that inhibits aggregation of
particles comprising from about 0.5 mol % to about 2 mol % of the total
lipid present in the particle.

[0019] In one preferred embodiment, the nucleic acid-lipid particle (e.g.,
SNALP) comprises: (a) an siRNA; (b) a cationic lipid comprising from
about 56.5 mol % to about 66.5 mol % of the total lipid present in the
particle; (c) cholesterol or a derivative thereof comprising from about
31.5 mol % to about 42.5 mol % of the total lipid present in the
particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to
about 2 mol % of the total lipid present in the particle. This preferred
embodiment of nucleic acid-lipid particle is generally referred to herein
as the "1:62" formulation.

[0020] In another preferred embodiment, the nucleic acid-lipid particle
(e.g., SNALP) comprises: (a) an siRNA; (b) a cationic lipid comprising
from about 52 mol % to about 62 mol % of the total lipid present in the
particle; (c) a mixture of a phospholipid and cholesterol or a derivative
thereof comprising from about 36 mol % to about 47 mol % of the total
lipid present in the particle; and (d) a PEG-lipid conjugate comprising
from about 1 mol % to about 2 mol % of the total lipid present in the
particle. This preferred embodiment of nucleic acid-lipid particle is
generally referred to herein as the "1:57" formulation.

[0022] In another aspect, the present invention provides methods for
introducing an active agent or therapeutic agent (e.g., nucleic acid)
into a cell, the method comprising contacting the cell with a lipid
particle described herein such as a nucleic acid-lipid particle (e.g.,
SNALP).

[0023] In yet another aspect, the present invention provides methods for
the in vivo delivery of an active agent or therapeutic agent (e.g.,
nucleic acid), the method comprising administering to a mammalian subject
a lipid particle described herein such as a nucleic acid-lipid particle
(e.g., SNALP).

[0024] In a further aspect, the present invention provides methods for
treating a disease or disorder in a mammalian subject in need thereof,
the method comprising administering to the mammalian subject a
therapeutically effective amount of a lipid particle described herein
such as a nucleic acid-lipid particle (e.g., SNALP).

[0025] Other objects, features, and advantages of the present invention
will be apparent to one of skill in the art from the following detailed
description and figures.

[0048] The present invention is based, in part, upon the surprising
discovery that lipid particles comprising from about 50 mol % to about 85
mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a
non-cationic lipid, and from about 0.5 mol % to about 2 mol % of a lipid
conjugate provide advantages when used for the in vitro or in vivo
delivery of an active agent, such as a therapeutic nucleic acid (e.g., an
interfering RNA). In particular, as illustrated by the Examples herein,
the present invention provides stable nucleic acid-lipid particles
(SNALP) that advantageously impart increased activity of the encapsulated
nucleic acid (e.g., an interfering RNA such as siRNA) and improved
tolerability of the formulations in vivo, resulting in a significant
increase in the therapeutic index as compared to nucleic acid-lipid
particle compositions previously described. Additionally, the SNALP of
the invention are stable in circulation, e.g., resistant to degradation
by nucleases in serum, and are substantially non-toxic to mammals such as
humans. As a non-limiting example, FIG. 3 of Example 4 shows that one
SNALP embodiment of the invention ("1:57 SNALP") was more than 10 times
as efficacious as compared to a nucleic acid-lipid particle previously
described ("2:30 SNALP") in mediating target gene silencing at a 10-fold
lower dose. Similarly, FIG. 2 of Example 3 shows that the "1:57 SNALP"
formulation was substantially more effective at silencing the expression
of a target gene as compared to nucleic acid-lipid particles previously
described ("2:40 SNALP").

[0049] In certain embodiments, the present invention provides improved
compositions for the delivery of interfering RNA such as siRNA molecules.
In particular, the Examples herein illustrate that the improved lipid
particle formulations of the invention are highly effective in
downregulating the mRNA and/or protein levels of target genes.
Furthermore, the Examples herein illustrate that the presence of certain
molar ratios of lipid components results in improved or enhanced activity
of these lipid particle formulations of the present invention. For
instance, the "1:57 SNALP" and "1:62 SNALP" formulations described herein
are exemplary formulations of the present invention that are particularly
advantageous because they provide improved efficacy and tolerability in
vivo, are serum-stable, are substantially non-toxic, are capable of
accessing extravascular sites, and are capable of reaching target cell
populations.

[0050] The lipid particles and compositions of the present invention may
be used for a variety of purposes, including the delivery of associated
or encapsulated therapeutic agents to cells, both in vitro and in vivo.
Accordingly, the present invention provides methods for treating diseases
or disorders in a subject in need thereof, by contacting the subject with
a lipid particle described herein comprising one or more suitable
therapeutic agents.

[0051] Various exemplary embodiments of the lipid particles of the
invention, as well as compositions and formulations comprising the same,
and their use to deliver therapeutic agents and modulate target gene and
protein expression, are described in further detail below.

II. Definitions

[0052] As used herein, the following terms have the meanings ascribed to
them unless specified otherwise.

[0053] The term "interfering RNA" or "RNAi" or "interfering RNA sequence"
refers to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA
(i.e., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that is capable of
reducing or inhibiting the expression of a target gene or sequence (e.g.,
by mediating the degradation or inhibiting the translation of mRNAs which
are complementary to the interfering RNA sequence) when the interfering
RNA is in the same cell as the target gene or sequence. Interfering RNA
thus refers to the single-stranded RNA that is complementary to a target
mRNA sequence or to the double-stranded RNA formed by two complementary
strands or by a single, self-complementary strand. Interfering RNA may
have substantial or complete identity to the target gene or sequence, or
may comprise a region of mismatch (i.e., a mismatch motif). The sequence
of the interfering RNA can correspond to the full-length target gene, or
a subsequence thereof.

[0054] Interfering RNA includes "small-interfering RNA" or "siRNA," e.g.,
interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in
length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides
in length, and is preferably about 20-24, 21-22, or 21-23 (duplex)
nucleotides in length (e.g., each complementary sequence of the
double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25
nucleotides in length, preferably about 20-24, 21-22, or 21-23
nucleotides in length, and the double-stranded siRNA is about 15-60,
15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably
about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes may
comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to
about 3 nucleotides and 5' phosphate termini. Examples of siRNA include,
without limitation, a double-stranded polynucleotide molecule assembled
from two separate stranded molecules, wherein one strand is the sense
strand and the other is the complementary antisense strand; a
double-stranded polynucleotide molecule assembled from a single stranded
molecule, where the sense and antisense regions are linked by a nucleic
acid-based or non-nucleic acid-based linker; a double-stranded
polynucleotide molecule with a hairpin secondary structure having
self-complementary sense and antisense regions; and a circular
single-stranded polynucleotide molecule with two or more loop structures
and a stem having self-complementary sense and antisense regions, where
the circular polynucleotide can be processed in vivo or in vitro to
generate an active double-stranded siRNA molecule.

[0056] As used herein, the term "mismatch motif" or "mismatch region"
refers to a portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA)
sequence that does not have 100% complementarity to its target sequence.
An interfering RNA may have at least one, two, three, four, five, six, or
more mismatch regions. The mismatch regions may be contiguous or may be
separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.
The mismatch motifs or regions may comprise a single nucleotide or may
comprise two, three, four, five, or more nucleotides.

[0057] An "effective amount" or "therapeutically effective amount" of an
active agent or therapeutic agent such as an interfering RNA is an amount
sufficient to produce the desired effect, e.g., an inhibition of
expression of a target sequence in comparison to the normal expression
level detected in the absence of an interfering RNA. Inhibition of
expression of a target gene or target sequence is achieved when the value
obtained with an interfering RNA relative to the control is about 90%,
85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target
gene or target sequence include, e.g., examination of protein or RNA
levels using techniques known to those of skill in the art such as dot
blots, northern blots, in situ hybridization, ELISA, immunoprecipitation,
enzyme function, as well as phenotypic assays known to those of skill in
the art.

[0058] By "decrease," "decreasing," "reduce," or "reducing" of an immune
response by an interfering RNA is intended to mean a detectable decrease
of an immune response to a given interfering RNA (e.g., a modified
interfering RNA). The amount of decrease of an immune response by a
modified interfering RNA may be determined relative to the level of an
immune response in the presence of an unmodified interfering RNA. A
detectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more
lower than the immune response detected in the presence of the unmodified
interfering RNA. A decrease in the immune response to interfering RNA is
typically measured by a decrease in cytokine production (e.g.,
IFNγ, IFNα, TNFα, IL-6, or IL-12) by a responder cell
in vitro or a decrease in cytokine production in the sera of a mammalian
subject after administration of the interfering RNA.

[0060] "Substantial identity" refers to a sequence that hybridizes to a
reference sequence under stringent conditions, or to a sequence that has
a specified percent identity over a specified region of a reference
sequence.

[0061] The phrase "stringent hybridization conditions" refers to
conditions under which a nucleic acid will hybridize to its target
sequence, typically in a complex mixture of nucleic acids, but to no
other sequences. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Probes,
"Overview of principles of hybridization and the strategy of nucleic acid
assays" (1993). Generally, stringent conditions are selected to be about
5-10° C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH, and nucleic concentration)
at which 50% of the probes complementary to the target hybridize to the
target sequence at equilibrium (as the target sequences are present in
excess, at Tm, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization.

[0062] Exemplary stringent hybridization conditions can be as follows: 50%
formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,
5×SSC, 1% SDS, incubating at 65° C., with wash in
0.2×SSC, and 0.1% SDS at 65° C. For PCR, a temperature of
about 36° C. is typical for low stringency amplification, although
annealing temperatures may vary between about 32° C. and
48° C. depending on primer length. For high stringency PCR
amplification, a temperature of about 62° C. is typical, although
high stringency annealing temperatures can range from about 50° C.
to about 65° C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency amplifications
include a denaturation phase of 90° C.-95° C. for 30 sec.-2
min., an annealing phase lasting 30 sec.-2 min., and an extension phase
of about 72° C. for 1-2 min. Protocols and guidelines for low and
high stringency amplification reactions are provided, e.g., in Innis et
al., PCR Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y. (1990).

[0063] Nucleic acids that do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides which
they encode are substantially identical. This occurs, for example, when a
copy of a nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code. In such cases, the nucleic acids typically
hybridize under moderately stringent hybridization conditions. Exemplary
"moderately stringent hybridization conditions" include a hybridization
in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a
wash in 1×SSC at 45° C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize that
alternative hybridization and wash conditions can be utilized to provide
conditions of similar stringency. Additional guidelines for determining
hybridization parameters are provided in numerous references, e.g.,
Current Protocols in Molecular Biology, Ausubel et al., eds.

[0064] The terms "substantially identical" or "substantial identity," in
the context of two or more nucleic acids, refer to two or more sequences
or subsequences that are the same or have a specified percentage of
nucleotides that are the same (i.e., at least about 60%, preferably at
least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a
specified region), when compared and aligned for maximum correspondence
over a comparison window, or designated region as measured using one of
the following sequence comparison algorithms or by manual alignment and
visual inspection. This definition, when the context indicates, also
refers analogously to the complement of a sequence. Preferably, the
substantial identity exists over a region that is at least about 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

[0065] For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary, and
sequence algorithm program parameters are designated. Default program
parameters can be used, or alternative parameters can be designated. The
sequence comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference sequence,
based on the program parameters.

[0066] A "comparison window," as used herein, includes reference to a
segment of any one of a number of contiguous positions selected from the
group consisting of from about 5 to about 60, usually about 10 to about
45, more usually about 15 to about 30, in which a sequence may be
compared to a reference sequence of the same number of contiguous
positions after the two sequences are optimally aligned. Methods of
alignment of sequences for comparison are well known in the art. Optimal
alignment of sequences for comparison can be conducted, e.g., by the
local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482
(1981), by the homology alignment algorithm of Needleman and Wunsch, J.
Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson
and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in
the Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds.
(1995 supplement)).

[0067] A preferred example of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.,
25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410
(1990), respectively. BLAST and BLAST 2.0 are used, with the parameters
described herein, to determine percent sequence identity for the nucleic
acids of the invention. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/).

[0068] The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul, Proc.
Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match between
two nucleotide sequences would occur by chance. For example, a nucleic
acid is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the reference
nucleic acid is less than about 0.2, more preferably less than about
0.01, and most preferably less than about 0.001.

[0069] The term "nucleic acid" as used herein refers to a polymer
containing at least two deoxyribonucleotides or ribonucleotides in either
single- or double-stranded form and includes DNA and RNA. DNA may be in
the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a
PCR product, vectors (P1, PAC, BAC, YAC, artificial chromosomes),
expression cassettes, chimeric sequences, chromosomal DNA, or derivatives
and combinations of these groups. RNA may be in the form of siRNA,
asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA,
tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include
nucleic acids containing known nucleotide analogs or modified backbone
residues or linkages, which are synthetic, naturally occurring, and
non-naturally occurring, and which have similar binding properties as the
reference nucleic acid. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and
peptide-nucleic acids (PNAs). Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides that have similar binding properties as the reference nucleic
acid. Unless otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions may be achieved by
generating sequences in which the third position of one or more selected
(or all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et
al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.
Probes, 8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA)
or ribose (RNA), a base, and a phosphate group. Nucleotides are linked
together through the phosphate groups. "Bases" include purines and
pyrimidines, which further include natural compounds adenine, thymine,
guanine, cytosine, uracil, inosine, and natural analogs, and synthetic
derivatives of purines and pyrimidines, which include, but are not
limited to, modifications which place new reactive groups such as, but
not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0070] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises partial length or entire length coding sequences
necessary for the production of a polypeptide or precursor polypeptide.

[0071] "Gene product," as used herein, refers to a product of a gene such
as an RNA transcript or a polypeptide.

[0072] The term "lipid" refers to a group of organic compounds that
include, but are not limited to, esters of fatty acids and are
characterized by being insoluble in water, but soluble in many organic
solvents. They are usually divided into at least three classes: (1)
"simple lipids," which include fats and oils as well as waxes; (2)
"compound lipids," which include phospholipids and glycolipids; and (3)
"derived lipids" such as steroids.

[0073] A "lipid particle" is used herein to refer to a lipid formulation
that can be used to deliver an active agent or therapeutic agent, such as
a nucleic acid (e.g., an interfering RNA), to a target site of interest.
In the lipid particle of the invention, which is typically formed from a
cationic lipid, a non-cationic lipid, and a conjugated lipid that
prevents aggregation of the particle, the active agent or therapeutic
agent may be encapsulated in the lipid, thereby protecting the agent from
enzymatic degradation.

[0074] As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle. A SNALP represents a particle made from lipids
(e.g., a cationic lipid, a non-cationic lipid, and a conjugated lipid
that prevents aggregation of the particle), wherein the nucleic acid
(e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA
(shRNA), dsRNA, or a plasmid, including plasmids from which an
interfering RNA is transcribed) is fully encapsulated within the lipid.
As used herein, the term "SNALP" includes an SPLP, which is the term used
to refer to a nucleic acid-lipid particle comprising a nucleic acid
(e.g., a plasmid) encapsulated within the lipid. SNALP and SPLP typically
contain a cationic lipid, a non-cationic lipid, and a lipid conjugate
(e.g., a PEG-lipid conjugate). SNALP and SPLP are extremely useful for
systemic applications, as they can exhibit extended circulation lifetimes
following intravenous (i.v.) injection, they can accumulate at distal
sites (e.g., sites physically separated from the administration site),
and they can mediate expression of the transfected gene or silencing of
target gene expression at these distal sites. SPLP include "pSPLP," which
comprise an encapsulated condensing agent-nucleic acid complex as set
forth in PCT Publication No. WO 00/03683, the disclosure of which is
herein incorporated by reference in its entirety for all purposes.

[0075] The lipid particles of the invention (e.g., SNALP) typically have a
mean diameter of from about 40 nm to about 150 nm, from about 50 nm to
about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about
110 nm, or from about 70 to about 90 nm, and are substantially non-toxic.
In addition, nucleic acids, when present in the lipid particles of the
invention, are resistant in aqueous solution to degradation with a
nuclease. Nucleic acid-lipid particles and their method of preparation
are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and
20070042031, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.

[0076] As used herein, "lipid encapsulated" can refer to a lipid particle
that provides an active agent or therapeutic agent, such as a nucleic
acid (e.g., an interfering RNA), with full encapsulation, partial
encapsulation, or both. In a preferred embodiment, the nucleic acid is
fully encapsulated in the lipid particle (e.g., to form an SPLP, pSPLP,
SNALP, or other nucleic acid-lipid particle).

[0077] The term "lipid conjugate" refers to a conjugated lipid that
inhibits aggregation of lipid particles. Such lipid conjugates include,
but are not limited to, polyamide oligomers (e.g., ATTA-lipid
conjugates), PEG-lipid conjugates, such as PEG coupled to
dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to
cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to
ceramides (see, e.g., U.S. Pat. No. 5,885,613, the disclosure of which is
herein incorporated by reference in its entirety for all purposes),
cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly
to the lipid or may be linked to the lipid via a linker moiety. Any
linker moiety suitable for coupling the PEG to a lipid can be used
including, e.g., non-ester containing linker moieties and
ester-containing linker moieties. In preferred embodiments, non-ester
containing linker moieties are used.

[0078] The term "amphipathic lipid" refers, in part, to any suitable
material wherein the hydrophobic portion of the lipid material orients
into a hydrophobic phase, while the hydrophilic portion orients toward
the aqueous phase. Hydrophilic characteristics derive from the presence
of polar or charged groups such as carbohydrates, phosphate, carboxylic,
sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
Hydrophobicity can be conferred by the inclusion of apolar groups that
include, but are not limited to, long-chain saturated and unsaturated
aliphatic hydrocarbon groups and such groups substituted by one or more
aromatic, cycloaliphatic, or heterocyclic group(s). Examples of
amphipathic compounds include, but are not limited to, phospholipids,
aminolipids, and sphingolipids.

[0079] Representative examples of phospholipids include, but are not
limited to, phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, phosphatidic acid,
palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and
dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,
such as sphingolipid, glycosphingolipid families, diacylglycerols, and
β-acyloxyacids, are also within the group designated as amphipathic
lipids. Additionally, the amphipathic lipids described above can be mixed
with other lipids including triglycerides and sterols.

[0080] The term "neutral lipid" refers to any of a number of lipid species
that exist either in an uncharged or neutral zwitterionic form at a
selected pH. At physiological pH, such lipids include, for example,
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,
sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0081] The term "non-cationic lipid" refers to any amphipathic lipid as
well as any other neutral lipid or anionic lipid.

[0082] The term "anionic lipid" refers to any lipid that is negatively
charged at physiological pH. These lipids include, but are not limited
to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,
diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,
N-succinyl phosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying
groups joined to neutral lipids.

[0083] The term "cationic lipid" refers to any of a number of lipid
species that carry a net positive charge at a selected pH, such as
physiological pH (e.g., pH of about 7.0). It has been surprisingly found
that cationic lipids comprising alkyl chains with multiple sites of
unsaturation, e.g., at least two or three sites of unsaturation, are
particularly useful for forming lipid particles with increased membrane
fluidity. A number of cationic lipids and related analogs, which are also
useful in the present invention, have been described in U.S. Patent
Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036;
5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT
Publication No. WO 96/10390, the disclosures of which are herein
incorporated by reference in their entirety for all purposes.
Non-limiting examples of cationic lipids are described in detail herein.
In some cases, the cationic lipids comprise a protonatable tertiary amine
(e.g., pH titratable) head group, C18 alkyl chains, ether linkages
between the head group and alkyl chains, and 0 to 3 double bonds. Such
lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA.

[0084] The term "hydrophobic lipid" refers to compounds having apolar
groups that include, but are not limited to, long-chain saturated and
unsaturated aliphatic hydrocarbon groups and such groups optionally
substituted by one or more aromatic, cycloaliphatic, or heterocyclic
group(s). Suitable examples include, but are not limited to,
diacylglycerol, dialkylglycerol, N-N-dialkylamino,
1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0085] The term "fusogenic" refers to the ability of a lipid particle,
such as a SNALP, to fuse with the membranes of a cell. The membranes can
be either the plasma membrane or membranes surrounding organelles, e.g.,
endosome, nucleus, etc.

[0086] As used herein, the term "aqueous solution" refers to a composition
comprising in whole, or in part, water.

[0087] As used herein, the term "organic lipid solution" refers to a
composition comprising in whole, or in part, an organic solvent having a
lipid.

[0088] "Distal site," as used herein, refers to a physically separated
site, which is not limited to an adjacent capillary bed, but includes
sites broadly distributed throughout an organism.

[0089] "Serum-stable" in relation to nucleic acid-lipid particles such as
SNALP means that the particle is not significantly degraded after
exposure to a serum or nuclease assay that would significantly degrade
free DNA or RNA. Suitable assays include, for example, a standard serum
assay, a DNAse assay, or an RNAse assay.

[0090] "Systemic delivery," as used herein, refers to delivery of lipid
particles that leads to a broad biodistribution of an active agent or
therapeutic agent such as an interfering RNA within an organism. Some
techniques of administration can lead to the systemic delivery of certain
agents, but not others. Systemic delivery means that a useful, preferably
therapeutic, amount of an agent is exposed to most parts of the body. To
obtain broad biodistribution generally requires a blood lifetime such
that the agent is not rapidly degraded or cleared (such as by first pass
organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before
reaching a disease site distal to the site of administration. Systemic
delivery of lipid particles can be by any means known in the art
including, for example, intravenous, subcutaneous, and intraperitoneal.
In a preferred embodiment, systemic delivery of lipid particles is by
intravenous delivery.

[0091] "Local delivery," as used herein, refers to delivery of an active
agent or therapeutic agent such as an interfering RNA directly to a
target site within an organism. For example, an agent can be locally
delivered by direct injection into a disease site such as a tumor or
other target site such as a site of inflammation or a target organ such
as the liver, heart, pancreas, kidney, and the like.

[0092] The term "mammal" refers to any mammalian species such as a human,
mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the
like.

[0093] The term "cancer" refers to any member of a class of diseases
characterized by the uncontrolled growth of aberrant cells. The term
includes all known cancers and neoplastic conditions, whether
characterized as malignant, benign, soft tissue, or solid, and cancers of
all stages and grades including pre- and post-metastatic cancers.
Examples of different types of cancer include, but are not limited to,
lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer,
small intestine cancer, stomach (gastric) cancer, esophageal cancer;
gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer,
breast cancer, ovarian cancer; cervical cancer, prostate cancer, renal
cancer (e.g., renal cell carcinoma), cancer of the central nervous
system, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and
neck cancers, osteogenic sarcomas, and blood cancers. Non-limiting
examples of specific types of liver cancer include hepatocellular
carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of
some other non-liver cancer cell type), and hepatoblastoma. As used
herein, a "tumor" comprises one or more cancerous cells.

III. Description of the Embodiments

[0094] The present invention provides novel, serum-stable lipid particles
comprising one or more active agents or therapeutic agents, methods of
making the lipid particles, and methods of delivering and/or
administering the lipid particles (e.g., for the treatment of a disease
or disorder).

[0095] In one aspect, the present invention provides lipid particles
comprising: (a) one or more active agents or therapeutic agents; (b) one
or more cationic lipids comprising from about 50 mol % to about 85 mol %
of the total lipid present in the particle; (c) one or more non-cationic
lipids comprising from about 13 mol % to about 49.5 mol % of the total
lipid present in the particle; and (d) one or more conjugated lipids that
inhibit aggregation of particles comprising from about 0.5 mol % to about
2 mol % of the total lipid present in the particle.

[0096] In certain embodiments, the active agent or therapeutic agent is
fully encapsulated within the lipid portion of the lipid particle such
that the active agent or therapeutic agent in the lipid particle is
resistant in aqueous solution to enzymatic degradation, e.g., by a
nuclease or protease. In certain other embodiments, the lipid particles
are substantially non-toxic to mammals such as humans.

[0097] In some embodiments, the active agent or therapeutic agent
comprises a nucleic acid. In certain instances, the nucleic acid
comprises an interfering RNA molecule such as, e.g., an siRNA, aiRNA,
miRNA, or mixtures thereof. In certain other instances, the nucleic acid
comprises single-stranded or double-stranded DNA, RNA, or a DNA/RNA
hybrid such as, e.g., an antisense oligonucleotide, a ribozyme, a
plasmid, an immunostimulatory oligonucleotide, or mixtures thereof.

[0098] In other embodiments, the active agent or therapeutic agent
comprises a peptide or polypeptide. In certain instances, the peptide or
polypeptide comprises an antibody such as, e.g., a polyclonal antibody, a
monoclonal antibody, an antibody fragment; a humanized antibody, a
recombinant antibody, a recombinant human antibody, a Primatized®
antibody, or mixtures thereof. In certain other instances, the peptide or
polypeptide comprises a cytokine, a growth factor, an apoptotic factor, a
differentiation-inducing factor, a cell-surface receptor, a ligand, a
hormone, a small molecule (e.g., small organic molecule or compound), or
mixtures thereof.

[0099] In preferred embodiments, the active agent or therapeutic agent
comprises an siRNA. In one embodiment, the siRNA molecule comprises a
double-stranded region of about 15 to about 60 nucleotides in length
(e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in
length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in
length). The siRNA molecules of the invention are capable of silencing
the expression of a target sequence in vitro and/or in vivo.

[0100] In some embodiments, the siRNA molecule comprises at least one
modified nucleotide. In certain preferred embodiments, the siRNA molecule
comprises one, two, three, four, five, six, seven, eight, nine, ten, or
more modified nucleotides in the double-stranded region. In certain
instances, the siRNA comprises from about 1% to about 100% (e.g., about
1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the
double-stranded region. In preferred embodiments, less than about 25%
(e.g., less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% to
about 25% (e.g., from about 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, or
10%-20%) of the nucleotides in the double-stranded region comprise
modified nucleotides.

[0102] The siRNA may comprise modified nucleotides in one strand (i.e.,
sense or antisense) or both strands of the double-stranded region of the
siRNA molecule. Preferably, uridine and/or guanosine nucleotides are
modified at selective positions in the double-stranded region of the
siRNA duplex. With regard to uridine nucleotide modifications, at least
one, two, three, four, five, six, or more of the uridine nucleotides in
the sense and/or antisense strand can be a modified uridine nucleotide
such as a 2'OMe-uridine nucleotide. In some embodiments, every uridine
nucleotide in the sense and/or antisense strand is a 2'OMe-uridine
nucleotide. With regard to guanosine nucleotide modifications, at least
one, two, three, four, five, six, or more of the guanosine nucleotides in
the sense and/or antisense strand can be a modified guanosine nucleotide
such as a 2'OMe-guanosine nucleotide. In some embodiments, every
guanosine nucleotide in the sense and/or antisense strand is a
2'OMe-guanosine nucleotide.

[0103] In certain embodiments, at least one, two, three, four, five, six,
seven, or more 5'-GU-3' motifs in an siRNA sequence may be modified,
e.g., by introducing mismatches to eliminate the 5'-GU-3' motifs and/or
by introducing modified nucleotides such as 2'OMe nucleotides. The
5'-GU-3' motif can be in the sense strand, the antisense strand, or both
strands of the siRNA sequence. The 5'-GU-3' motifs may be adjacent to
each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, or more nucleotides.

[0104] In some preferred embodiments, a modified siRNA molecule is less
immunostimulatory than a corresponding unmodified siRNA sequence. In such
embodiments, the modified siRNA molecule with reduced immunostimulatory
properties advantageously retains RNAi activity against the target
sequence. In another embodiment, the immunostimulatory properties of the
modified siRNA molecule and its ability to silence target gene expression
can be balanced or optimized by the introduction of minimal and selective
2'OMe modifications within the siRNA sequence such as, e.g., within the
double-stranded region of the siRNA duplex. In certain instances, the
modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than the
corresponding unmodified siRNA. It will be readily apparent to those of
skill in the art that the immunostimulatory properties of the modified
siRNA molecule and the corresponding unmodified siRNA molecule can be
determined by, for example, measuring INF-α and/or IL-6 levels from
about two to about twelve hours after systemic administration in a mammal
or transfection of a mammalian responder cell using an appropriate
lipid-based delivery system (such as the SNALP delivery system disclosed
herein).

[0105] In certain embodiments, a modified siRNA molecule has an IC50
(i.e., half-maximal inhibitory concentration) less than or equal to
ten-fold that of the corresponding unmodified siRNA (i.e., the modified
siRNA has an IC50 that is less than or equal to ten-times the
IC50 of the corresponding unmodified siRNA). In other embodiments,
the modified siRNA has an IC50 less than or equal to three-fold that
of the corresponding unmodified siRNA sequence. In yet other embodiments,
the modified siRNA has an IC50 less than or equal to two-fold that
of the corresponding unmodified siRNA. It will be readily apparent to
those of skill in the art that a dose-response curve can be generated and
the IC50 values for the modified siRNA and the corresponding
unmodified siRNA can be readily determined using methods known to those
of skill in the art.

[0107] In some embodiments, the siRNA molecule does not comprise phosphate
backbone modifications, e.g., in the sense and/or antisense strand of the
double-stranded region. In other embodiments, the siRNA comprises one,
two, three, four, or more phosphate backbone modifications, e.g., in the
sense and/or antisense strand of the double-stranded region. In preferred
embodiments, the siRNA does not comprise phosphate backbone
modifications.

[0108] In further embodiments, the siRNA does not comprise 2'-deoxy
nucleotides, e.g., in the sense and/or antisense strand of the
double-stranded region. In yet further embodiments, the siRNA comprises
one, two, three, four, or more 2'-deoxy nucleotides, e.g., in the sense
and/or antisense strand of the double-stranded region. In preferred
embodiments, the siRNA does not comprise 2'-deoxy nucleotides.

[0109] In certain instances, the nucleotide at the 3'-end of the
double-stranded region in the sense and/or antisense strand is not a
modified nucleotide. In certain other instances, the nucleotides near the
3'-end (e.g., within one, two, three, or four nucleotides of the 3'-end)
of the double-stranded region in the sense and/or antisense strand are
not modified nucleotides.

[0110] The siRNA molecules described herein may have 3' overhangs of one,
two, three, four, or more nucleotides on one or both sides of the
double-stranded region, or may lack overhangs (i.e., have blunt ends) on
one or both sides of the double-stranded region. Preferably, the siRNA
has 3' overhangs of two nucleotides on each side of the double-stranded
region. In certain instances, the 3' overhang on the antisense strand has
complementarity to the target sequence and the 3' overhang on the sense
strand has complementarity to a complementary strand of the target
sequence. Alternatively, the 3' overhangs do not have complementarity to
the target sequence or the complementary strand thereof. In some
embodiments, the 3' overhangs comprise one, two, three, four, or more
nucleotides such as 2'-deoxy(2'H) nucleotides. In certain preferred
embodiments, the 3' overhangs comprise deoxythymidine (dT) and/or uridine
nucleotides. In other embodiments, one or more of the nucleotides in the
3' overhangs on one or both sides of the double-stranded region comprise
modified nucleotides. Non-limiting examples of modified nucleotides are
described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides,
2'-deoxy nucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and
mixtures thereof. In preferred embodiments, one, two, three, four, or
more nucleotides in the 3' overhangs present on the sense and/or
antisense strand of the siRNA comprise 2'OMe nucleotides (e.g., 2'OMe
purine and/or pyrimidine nucleotides) such as, for example,
2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine
nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof.

[0111] The siRNA may comprise at least one or a cocktail (e.g., at least
two, three, four, five, six, seven, eight, nine, ten, or more) of
unmodified and/or modified siRNA sequences that silence target gene
expression. The cocktail of siRNA may comprise sequences which are
directed to the same region or domain (e.g., a "hot spot") and/or to
different regions or domains of one or more target genes. In certain
instances, one or more (e.g., at least two, three, four, five, six,
seven, eight, nine, ten, or more) modified siRNA that silence target gene
expression are present in a cocktail. In certain other instances, one or
more (e.g., at least two, three, four, five, six, seven, eight, nine,
ten, or more) unmodified siRNA sequences that silence target gene
expression are present in a cocktail.

[0112] In some embodiments, the antisense strand of the siRNA molecule
comprises or consists of a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a
portion thereof. In other embodiments, the antisense strand of the siRNA
molecule comprises or consists of a sequence that is 100% complementary
to the target sequence or a portion thereof. In further embodiments, the
antisense strand of the siRNA molecule comprises or consists of a
sequence that specifically hybridizes to the target sequence or a portion
thereof.

[0113] In further embodiments, the sense strand of the siRNA molecule
comprises or consists of a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion
thereof. In additional embodiments, the sense strand of the siRNA
molecule comprises or consists of a sequence that is 100% identical to
the target sequence or a portion thereof.

[0115] The synthesis of cationic lipids such as DLin-K-C2-DMA ("XTC2"),
DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as
additional cationic lipids, is described in U.S. Provisional Application
No. 61/104,212, filed Oct. 9, 2008, the disclosure of which is herein
incorporated by reference in its entirety for all purposes. The synthesis
of cationic lipids such as DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA,
DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ,
DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is
described in PCT Application No. PCT/US08/88676, filed Dec. 31, 2008, the
disclosure of which is herein incorporated by reference in its entirety
for all purposes. The synthesis of cationic lipids such as CLinDMA, as
well as additional cationic lipids, is described in U.S. Patent
Publication No. 20060240554, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.

[0116] In some embodiments, the cationic lipid may comprise from about 50
mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from
about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %,
from about 50 mol % to about 70 mol %, from about 50 mol % to about 65
mol %, or from about 50 mol % to about 60 mol % of the total lipid
present in the particle.

[0117] In other embodiments, the cationic lipid may comprise from about 55
mol % to about 90 mol %, from about 55 mol % to about 85 mol %, from
about 55 mol % to about 80 mol %, from about 55 mol % to about 75 mol %,
from about 55 mol % to about 70 mol %, or from about 55 mol % to about 65
mol % of the total lipid present in the particle.

[0118] In yet other embodiments, the cationic lipid may comprise from
about 60 mol % to about 90 mol %, from about 60 mol % to about 85 mol %,
from about 60 mol % to about 80 mol %, from about 60 mol % to about 75
mol %, or from about 60 mol % to about 70 mol % of the total lipid
present in the particle.

[0119] In still yet other embodiments, the cationic lipid may comprise
from about 65 mol % to about 90 mol %, from about 65 mol % to about 85
mol %, from about 65 mol % to about 80 mol %, or from about 65 mol % to
about 75 mol % of the total lipid present in the particle.

[0120] In further embodiments, the cationic lipid may comprise from about
70 mol % to about 90 mol %, from about 70 mol % to about 85 mol %, from
about 70 mol % to about 80 mol %, from about 75 mol % to about 90 mol %,
from about 75 mol % to about 85 mol %, or from about 80 mol % to about 90
mol % of the total lipid present in the particle.

[0122] In the lipid particles of the invention (e.g., SNALP comprising an
interfering RNA such as siRNA), the non-cationic lipid may comprise,
e.g., one or more anionic lipids and/or neutral lipids. In preferred
embodiments, the non-cationic lipid comprises one of the following
neutral lipid components: (1) cholesterol or a derivative thereof; (2) a
phospholipid; or (3) a mixture of a phospholipid and cholesterol or a
derivative thereof.

[0123] Examples of cholesterol derivatives include, but are not limited
to, cholestanol, cholestanone, cholestenone, coprostanol,
cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and
mixtures thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether is
described herein.

[0125] In some embodiments, the non-cationic lipid (e.g., one or more
phospholipids and/or cholesterol) may comprise from about 10 mol % to
about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol
% to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30
mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from
about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %,
from about 25 mol % to about 55 mol %, from about 30 mol % to about 55
mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to
about 50 mol % or from about 20 mol % to about 50 mol % of the total
lipid present in the particle. When the non-cationic lipid is a mixture
of a phospholipid and cholesterol or a cholesterol derivative, the
mixture may comprise up to about 40, 50, or 60 mol % of the total lipid
present in the particle.

[0126] In other embodiments, the non-cationic lipid (e.g., one or more
phospholipids and/or cholesterol) may comprise from about 10 mol % to
about 49.5 mol %, from about 13 mol % to about 49.5 mol %, from about 15
mol % to about 49.5 mol %, from about 20 mol % to about 49.5 mol %, from
about 25 mol % to about 49.5 mol %, from about 30 mol % to about 49.5 mol
%, from about 35 mol % to about 49.5 mol %, or from about 40 mol % to
about 49.5 mol % of the total lipid present in the particle.

[0127] In yet other embodiments, the non-cationic lipid (e.g., one or more
phospholipids and/or cholesterol) may comprise from about 10 mol % to
about 45 mol %, from about 13 mol % to about 45 mol %, from about 15 mol
% to about 45 mol %, from about 20 mol % to about 45 mol %, from about 25
mol % to about 45 mol %, from about 30 mol % to about 45 mol %, or from
about 35 mol % to about 45 mol % of the total lipid present in the
particle.

[0128] In still yet other embodiments, the non-cationic lipid (e.g., one
or more phospholipids and/or cholesterol) may comprise from about 10 mol
% to about 40 mol %, from about 13 mol % to about 40 mol %, from about 15
mol % to about 40 mol %, from about 20 mol % to about 40 mol %, from
about 25 mol % to about 40 mol %, or from about 30 mol % to about 40 mol
% of the total lipid present in the particle.

[0129] In further embodiments, the non-cationic lipid (e.g., one or more
phospholipids and/or cholesterol) may comprise from about 10 mol % to
about 35 mol %, from about 13 mol % to about 35 mol %, from about 15 mol
% to about 35 mol %, from about 20 mol % to about 35 mol %, or from about
25 mol % to about 35 mol % of the total lipid present in the particle.

[0130] In yet further embodiments, the non-cationic lipid (e.g., one or
more phospholipids and/or cholesterol) may comprise from about 10 mol %
to about 30 mol %, from about 13 mol % to about 30 mol %, from about 15
mol % to about 30 mol %, from about 20 mol % to about 30 mol %, from
about 10 mol % to about 25 mol %, from about 13 mol % to about 25 mol %,
or from about 15 mol % to about 25 mol % of the total lipid present in
the particle.

[0132] In certain preferred embodiments, the non-cationic lipid comprises
cholesterol or a derivative thereof of from about 31.5 mol % to about
42.5 mol % of the total lipid present in the particle. As a non-limiting
example, a phospholipid-free lipid particle of the invention may comprise
cholesterol or a derivative thereof at about 37 mol % of the total lipid
present in the particle. In other preferred embodiments, a
phospholipid-free lipid particle of the invention may comprise
cholesterol or a derivative thereof of from about 30 mol % to about 45
mol %, from about 30 mol % to about 40 mol %, from about 30 mol % to
about 35 mol %, from about 35 mol % to about 45 mol %, from about 40 mol
% to about 45 mol %, from about 32 mol % to about 45 mol %, from about 32
mol % to about 42 mol %, from about 32 mol % to about 40 mol %, from
about 34 mol % to about 45 mol %, from about 34 mol % to about 42 mol %,
from about 34 mol % to about 40 mol %, or about 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereof
or range therein) of the total lipid present in the particle.

[0133] In certain other preferred embodiments, the non-cationic lipid
comprises a mixture of: (i) a phospholipid of from about 4 mol % to about
10 mol % of the total lipid present in the particle; and (ii) cholesterol
or a derivative thereof of from about 30 mol % to about 40 mol % of the
total lipid present in the particle. As a non-limiting example, a lipid
particle comprising a mixture of a phospholipid and cholesterol may
comprise DPPC at about 7 mol % and cholesterol at about 34 mol % of the
total lipid present in the particle. In other embodiments, the
non-cationic lipid comprises a mixture of: (i) a phospholipid of from
about 3 mol % to about 15 mol %, from about 4 mol % to about 15 mol %,
from about 4 mol % to about 12 mol %, from about 4 mol % to about 10 mol
%, from about 4 mol % to about 8 mol %, from about 5 mol % to about 12
mol %, from about 5 mol % to about 9 mol %, from about 6 mol % to about
12 mol %, from about 6 mol % to about 10 mol %, or about 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15 mol % (or any fraction thereof or range
therein) of the total lipid present in the particle; and (ii) cholesterol
or a derivative thereof of from about 25 mol % to about 45 mol %, from
about 30 mol % to about 45 mol %, from about 25 mol % to about 40 mol %,
from about 30 mol % to about 40 mol %, from about 25 mol % to about 35
mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to
about 45 mol %, from about 40 mol % to about 45 mol %, from about 28 mol
% to about 40 mol %, from about 28 mol % to about 38 mol %, from about 30
mol % to about 38 mol %, from about 32 mol % to about 36 mol %, or about
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, or 45 mol % (or any fraction thereof or range therein) of the
total lipid present in the particle.

[0134] In further preferred embodiments, the non-cationic lipid comprises
a mixture of: (i) a phospholipid of from about 10 mol % to about 30 mol %
of the total lipid present in the particle; and (ii) cholesterol or a
derivative thereof of from about 10 mol % to about 30 mol % of the total
lipid present in the particle. As a non-limiting example, a lipid
particle comprising a mixture of a phospholipid and cholesterol may
comprise DPPC at about 20 mol % and cholesterol at about 20 mol % of the
total lipid present in the particle. In other embodiments, the
non-cationic lipid comprises a mixture of: (i) a phospholipid of from
about 10 mol % to about 30 mol %, from about 10 mol % to about 25 mol %,
from about 10 mol % to about 20 mol %, from about 15 mol % to about 30
mol %, from about 20 mol % to about 30 mol %, from about 15 mol % to
about 25 mol %, from about 12 mol % to about 28 mol %, from about 14 mol
% to about 26 mol %, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol % (or any fraction thereof
or range therein) of the total lipid present in the particle; and (ii)
cholesterol or a derivative thereof of from about 10 mol % to about 30
mol %, from about 10 mol % to about 25 mol %, from about 10 mol % to
about 20 mol %, from about 15 mol % to about 30 mol %, from about 20 mol
% to about 30 mol %, from about 15 mol % to about 25 mol %, from about 12
mol % to about 28 mol %, from about 14 mol % to about 26 mol %, or about
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 mol % (or any fraction thereof or range therein) of the
total lipid present in the particle.

[0135] In the lipid particles of the invention (e.g., SNALP comprising an
interfering RNA such as siRNA), the conjugated lipid that inhibits
aggregation of particles may comprise, e.g., one or more of the
following: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide
(ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), or
mixtures thereof. In one preferred embodiment, the nucleic acid-lipid
particles comprise either a PEG-lipid conjugate or an ATTA-lipid
conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid
conjugate is used together with a CPL. The conjugated lipid that inhibits
aggregation of particles may comprise a PEG-lipid including, e.g., a
PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a
PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA
conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl
(C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18),
or mixtures thereof.

[0136] Additional PEG-lipid conjugates suitable for use in the invention
include, but are not limited to,
mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The
synthesis of PEG-C-DOMG is described in PCT Application No.
PCT/US08/88676, filed Dec. 31, 2008, the disclosure of which is herein
incorporated by reference in its entirety for all purposes. Yet
additional PEG-lipid conjugates suitable for use in the invention
include, without limitation,
1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbam-
oyl-ω-methyl-poly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2
KPEG-DMG is described in U.S. Pat. No. 7,404,969, the disclosure of which
is herein incorporated by reference in its entirety for all purposes.

[0137] The PEG moiety of the PEG-lipid conjugates described herein may
comprise an average molecular weight ranging from about 550 daltons to
about 10,000 daltons. In certain instances, the PEG moiety has an average
molecular weight of from about 750 daltons to about 5,000 daltons (e.g.,
from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons
to about 3,000 daltons, from about 750 daltons to about 3,000 daltons,
from about 750 daltons to about 2,000 daltons, etc.). In preferred
embodiments, the PEG moiety has an average molecular weight of about
2,000 daltons or about 750 daltons.

[0138] In some embodiments, the conjugated lipid that inhibits aggregation
of particles is a CPL that has the formula: A-W-Y, wherein A is a lipid
moiety, W is a hydrophilic polymer, and Y is a polycationic moiety. W may
be a polymer selected from the group consisting of polyethyleneglycol
(PEG), polyamide, polylactic acid, polyglycolic acid, polylactic
acid/polyglycolic acid copolymers, or combinations thereof, the polymer
having a molecular weight of from about 250 to about 7000 daltons. In
some embodiments, Y has at least 4 positive charges at a selected pH. In
some embodiments, Y may be lysine, arginine, asparagine, glutamine,
derivatives thereof, or combinations thereof.

[0139] In certain instances, the conjugated lipid that inhibits
aggregation of particles (e.g., PEG-lipid conjugate) may comprise from
about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %,
from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9
mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to
about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2
mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from
about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol
%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol %
(or any fraction thereof or range therein) of the total lipid present in
the particle.

[0140] In the lipid particles of the invention, the active agent or
therapeutic agent may be fully encapsulated within the lipid portion of
the particle, thereby protecting the active agent or therapeutic agent
from enzymatic degradation. In preferred embodiments, a SNALP comprising
a nucleic acid such as an interfering RNA (e.g., siRNA) is fully
encapsulated within the lipid portion of the particle, thereby protecting
the nucleic acid from nuclease degradation. In certain instances, the
nucleic acid in the SNALP is not substantially degraded after exposure of
the particle to a nuclease at 37° C. for at least about 20, 30,
45, or 60 minutes. In certain other instances, the nucleic acid in the
SNALP is not substantially degraded after incubation of the particle in
serum at 37° C. for at least about 30, 45, or 60 minutes or at
least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34, or 36 hours. In other embodiments, the active agent or
therapeutic agent (e.g., nucleic acid such as siRNA) is complexed with
the lipid portion of the particle. One of the benefits of the
formulations of the present invention is that the lipid particle
compositions are substantially non-toxic to mammals such as humans.

[0141] The term "fully encapsulated" indicates that the active agent or
therapeutic agent in the lipid particle is not significantly degraded
after exposure to serum or a nuclease or protease assay that would
significantly degrade free DNA, RNA, or protein. In a fully encapsulated
system, preferably less than about 25% of the active agent or therapeutic
agent in the particle is degraded in a treatment that would normally
degrade 100% of free active agent or therapeutic agent, more preferably
less than about 10%, and most preferably less than about 5% of the active
agent or therapeutic agent in the particle is degraded. In the context of
nucleic acid therapeutic agents, full encapsulation may be determined by
an Oligreen® assay. Oligreen® is an ultra-sensitive fluorescent
nucleic acid stain for quantitating oligonucleotides and single-stranded
DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad,
Calif.). "Fully encapsulated" also indicates that the lipid particles are
serum-stable, that is, that they do not rapidly decompose into their
component parts upon in vivo administration.

[0142] In another aspect, the present invention provides a lipid particle
(e.g., SNALP) composition comprising a plurality of lipid particles. In
preferred embodiments, the active agent or therapeutic agent (e.g.,
nucleic acid) is fully encapsulated within the lipid portion of the lipid
particles (e.g., SNALP), such that from about 30% to about 100%, from
about 40% to about 100%, from about 50% to about 100%, from about 60% to
about 100%, from about 70% to about 100%, from about 80% to about 100%,
from about 90% to about 100%, from about 30% to about 95%, from about 40%
to about 95%, from about 50% to about 95%, from about 60% to about 95%,
%, from about 70% to about 95%, from about 80% to about 95%, from about
85% to about 95%, from about 90% to about 95%, from about 30% to about
90%, from about 40% to about 90%, from about 50% to about 90%, from about
60% to about 90%, from about 70% to about 90%, from about 80% to about
90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any
fraction thereof or range therein) of the lipid particles (e.g., SNALP)
have the active agent or therapeutic agent encapsulated therein.

[0143] Typically, the lipid particles (e.g., SNALP) of the invention have
a lipid:active agent (e.g., lipid:nucleic acid) ratio (mass/mass ratio)
of from about 1 to about 100. In some instances, the lipid:active agent
(e.g., lipid:nucleic acid) ratio (mass/mass ratio) ranges from about 1 to
about 50, from about 2 to about 25, from about 3 to about 20, from about
4 to about 15, or from about 5 to about 10. In preferred embodiments, the
lipid particles of the invention have a lipid:active agent (e.g.,
lipid:nucleic acid) ratio (mass/mass ratio) of from about 5 to about 15,
e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fraction
thereof or range therein).

[0144] Typically, the lipid particles (e.g., SNALP) of the invention have
a mean diameter of from about 40 nm to about 150 nm. In preferred
embodiments, the lipid particles (e.g., SNALP) of the invention have a
mean diameter of from about 40 nm to about 130 nm, from about 40 nm to
about 120 nm, from about 40 nm to about 100 nm, from about 50 nm to about
120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 120
nm, from about 60 nm to about 110 nm, from about 60 nm to about 100 nm,
from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from
about 70 nm to about 120 nm, from about 70 nm to about 110 nm, from about
70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm
to about 80 nm, or less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80
nm (or any fraction thereof or range therein).

[0145] In one specific embodiment of the invention, the SNALP comprises:
(a) one or more unmodified and/or modified interfering RNA (e.g., siRNA,
aiRNA, miRNA) that silence target gene expression; (b) a cationic lipid
comprising from about 56.5 mol % to about 66.5 mol % of the total lipid
present in the particle; (c) a non-cationic lipid comprising from about
31.5 mol % to about 42.5 mol % of the total lipid present in the
particle; and (d) a conjugated lipid that inhibits aggregation of
particles comprising from about 1 mol % to about 2 mol % of the total
lipid present in the particle. This specific embodiment of SNALP is
generally referred to herein as the "1:62" formulation. In a preferred
embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA ("XTC2"), the
non-cationic lipid is cholesterol, and the conjugated lipid is a PEG-DAA
conjugate. Although these are preferred embodiments of the 1:62
formulation, those of skill in the art will appreciate that other
cationic lipids, non-cationic lipids (including other cholesterol
derivatives), and conjugated lipids can be used in the 1:62 formulation
as described herein.

[0146] In another specific embodiment of the invention, the SNALP
comprises: (a) one or more unmodified and/or modified interfering RNA
(e.g., siRNA, aiRNA, miRNA) that silence target gene expression; (b) a
cationic lipid comprising from about 52 mol % to about 62 mol % of the
total lipid present in the particle; (c) a non-cationic lipid comprising
from about 36 mol % to about 47 mol % of the total lipid present in the
particle; and (d) a conjugated lipid that inhibits aggregation of
particles comprising from about 1 mol % to about 2 mol % of the total
lipid present in the particle. This specific embodiment of SNALP is
generally referred to herein as the "1:57" formulation. In one preferred
embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA ("XTC2"), the
non-cationic lipid is a mixture of a phospholipid (such as DPPC) and
cholesterol, wherein the phospholipid comprises from about 5 mol % to
about 9 mol % of the total lipid present in the particle (e.g., about 7.1
mol %) and the cholesterol (or cholesterol derivative) comprises from
about 32 mol % to about 37 mol % of the total lipid present in the
particle (e.g., about 34.3 mol %), and the PEG-lipid is a PEG-DAA (e.g.,
PEG-cDMA). In another preferred embodiment, the cationic lipid is DLinDMA
or DLin-K-C2-DMA ("XTC2"), the non-cationic lipid is a mixture of a
phospholipid (such as DPPC) and cholesterol, wherein the phospholipid
comprises from about 15 mol % to about 25 mol % of the total lipid
present in the particle (e.g., about 20 mol %) and the cholesterol (or
cholesterol derivative) comprises from about 15 mol % to about 25 mol %
of the total lipid present in the particle (e.g., about 20 mol %), and
the PEG-lipid is a PEG-DAA (e.g., PEG-cDMA). Although these are preferred
embodiments of the 1:57 formulation, those of skill in the art will
appreciate that other cationic lipids, non-cationic lipids (including
other phospholipids and other cholesterol derivatives), and conjugated
lipids can be used in the 1:57 formulation as described herein.

[0147] In preferred embodiments, the 1:62 SNALP formulation is a
three-component system which is phospholipid-free and comprises about 1.5
mol % PEG-cDMA (or PEG-cDSA), about 61.5 mol % DLinDMA (or XTC2), and
about 36.9 mol % cholesterol (or derivative thereof). In other preferred
embodiments, the 1:57 SNALP formulation is a four-component system which
comprises about 1.4 mol % PEG-cDMA (or PEG-cDSA), about 57.1 mol %
DLinDMA (or XTC2), about 7.1 mol % DPPC, and about 34.3 mol % cholesterol
(or derivative thereof). In yet other preferred embodiments, the 1:57
SNALP formulation is a four-component system which comprises about 1.4
mol % PEG-cDMA (or PEG-cDSA), about 57.1 mol % DLinDMA (or XTC2), about
20 mol % DPPC, and about 20 mol % cholesterol (or derivative thereof). It
should be understood that these SNALP formulations are target
formulations, and that the amount of lipid (both cationic and
non-cationic) present and the amount of lipid conjugate present in the
SNALP formulations may vary.

[0148] The present invention also provides a pharmaceutical composition
comprising a lipid particle (e.g., SNALP) described herein and a
pharmaceutically acceptable carrier.

[0149] In a further aspect, the present invention provides a method for
introducing one or more active agents or therapeutic agents (e.g.,
nucleic acid) into a cell, comprising contacting the cell with a lipid
particle (e.g., SNALP) described herein. In one embodiment, the cell is
in a mammal and the mammal is a human. In another embodiment, the present
invention provides a method for the in vivo delivery of one or more
active agents or therapeutic agents (e.g., nucleic acid), comprising
administering to a mammalian subject a lipid particle (e.g., SNALP)
described herein. In a preferred embodiment, the mode of administration
includes, but is not limited to, oral, intranasal, intravenous,
intraperitoneal, intramuscular, intra-articular, intralesional,
intratracheal, subcutaneous, and intradermal. Preferably, the mammalian
subject is a human.

[0150] In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the
total injected dose of the lipid particles (e.g., SNALP) is present in
plasma about 8, 12, 24, 36, or 48 hours after injection. In other
embodiments, more than about 20%, 30%, 40% and as much as about 60%, 70%
or 80% of the total injected dose of the lipid particles (e.g., SNALP) is
present in plasma about 8, 12, 24, 36, or 48 hours after injection. In
certain instances, more than about 10% of a plurality of the particles is
present in the plasma of a mammal about 1 hour after administration. In
certain other instances, the presence of the lipid particles (e.g.,
SNALP) is detectable at least about 1 hour after administration of the
particle. In certain embodiments, the presence of an active agent or
therapeutic agent such as an interfering RNA (e.g., siRNA) is detectable
in cells of the lung, liver, tumor, or at a site of inflammation at about
8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In other
embodiments, downregulation of expression of a target sequence by an
active agent or therapeutic agent such as an interfering RNA (e.g.,
siRNA) is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after
administration. In yet other embodiments, downregulation of expression of
a target sequence by an active agent or therapeutic agent such as an
interfering RNA (e.g., siRNA) occurs preferentially in tumor cells or in
cells at a site of inflammation. In further embodiments, the presence or
effect of an active agent or therapeutic agent such as an interfering RNA
(e.g., siRNA) in cells at a site proximal or distal to the site of
administration or in cells of the lung, liver, or a tumor is detectable
at about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16,
18, 19, 20, 22, 24, 26, or 28 days after administration. In additional
embodiments, the lipid particles (e.g., SNALP) of the invention are
administered parenterally or intraperitoneally.

[0151] In some embodiments, the lipid particles (e.g., SNALP) of the
invention are particularly useful in methods for the therapeutic delivery
of one or more nucleic acids comprising an interfering RNA sequence
(e.g., siRNA). In particular, it is an object of this invention to
provide in vitro and in vivo methods for treatment of a disease or
disorder in a mammal (e.g., a rodent such as a mouse or a primate such as
a human, chimpanzee, or monkey) by downregulating or silencing the
transcription and/or translation of one or more target nucleic acid
sequences or genes of interest. As a non-limiting example, the methods of
the invention are useful for in vivo delivery of interfering RNA (e.g.,
siRNA) to the liver and/or tumor of a mammalian subject. In certain
embodiments, the disease or disorder is associated with expression and/or
overexpression of a gene and expression or overexpression of the gene is
reduced by the interfering RNA (e.g., siRNA). In certain other
embodiments, a therapeutically effective amount of the lipid particle
(e.g., SNALP) may be administered to the mammal. In some instances, an
interfering RNA (e.g., siRNA) is formulated into a SNALP, and the
particles are administered to patients requiring such treatment. In other
instances, cells are removed from a patient, the interfering RNA (e.g.,
siRNA) is delivered in vitro (e.g., using a SNALP described herein), and
the cells are reinjected into the patient.

[0152] In an additional aspect, the present invention provides lipid
particles (e.g., SNALP) comprising asymmetrical interfering RNA (aiRNA)
molecules that silence the expression of a target gene and methods of
using such particles to silence target gene expression.

[0153] In one embodiment, the aiRNA molecule comprises a double-stranded
(duplex) region of about 10 to about 25 (base paired) nucleotides in
length, wherein the aiRNA molecule comprises an antisense strand
comprising 5' and 3' overhangs, and wherein the aiRNA molecule is capable
of silencing target gene expression.

[0154] In certain instances, the aiRNA molecule comprises a
double-stranded (duplex) region of about 12-20, 12-19, 12-18, 13-17, or
14-17 (base paired) nucleotides in length, more typically 12, 13, 14, 15,
16, 17, 18, 19, or 20 (base paired) nucleotides in length. In certain
other instances, the 5' and 3' overhangs on the antisense strand comprise
sequences that are complementary to the target RNA sequence, and may
optionally further comprise nontargeting sequences. In some embodiments,
each of the 5' and 3' overhangs on the antisense strand comprises or
consists of one, two, three, four, five, six, seven, or more nucleotides.

[0155] In other embodiments, the aiRNA molecule comprises modified
nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F
nucleotides, 2'-deoxy nucleotides, 2'-O-MOE nucleotides, LNA nucleotides,
and mixtures thereof. In a preferred embodiment, the aiRNA molecule
comprises 2'OMe nucleotides. As a non-limiting example, the 2'OMe
nucleotides may be selected from the group consisting of 2'OMe-guanosine
nucleotides, 2'OMe-uridine nucleotides, and mixtures thereof.

[0156] In a related aspect, the present invention provides lipid particles
(e.g., SNALP) comprising microRNA (miRNA) molecules that silence the
expression of a target gene and methods of using such compositions to
silence target gene expression.

[0157] In one embodiment, the miRNA molecule comprises about 15 to about
60 nucleotides in length, wherein the miRNA molecule is capable of
silencing target gene expression.

[0158] In certain instances, the miRNA molecule comprises about 15-50,
15-40, or 15-30 nucleotides in length, more typically about 15-25 or
19-25 nucleotides in length, and are preferably about 20-24, 21-22, or
21-23 nucleotides in length. In a preferred embodiment, the miRNA
molecule is a mature miRNA molecule targeting an RNA sequence of
interest.

[0159] In some embodiments, the miRNA molecule comprises modified
nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F
nucleotides, 2'-deoxy nucleotides, 2'-O-MOE nucleotides, LNA nucleotides,
and mixtures thereof. In a preferred embodiment, the miRNA molecule
comprises 2'OMe nucleotides. As a non-limiting example, the 2'OMe
nucleotides may be selected from the group consisting of 2'OMe-guanosine
nucleotides, 2'OMe-uridine nucleotides, and mixtures thereof.

[0160] As such, the lipid particles of the invention (e.g., SNALP) are
advantageous and suitable for use in the administration of active agents
or therapeutic agents such as nucleic acid (e.g., interfering RNA such as
siRNA, aiRNA, and/or miRNA) to a subject (e.g., a mammal such as a human)
because they are stable in circulation, of a size required for
pharmacodynamic behavior resulting in access to extravascular sites, and
are capable of reaching target cell populations.

IV. Active Agents

[0161] Active agents (e.g., therapeutic agents) include any molecule or
compound capable of exerting a desired effect on a cell, tissue, organ,
or subject. Such effects may be, e.g., biological, physiological, and/or
cosmetic. Active agents may be any type of molecule or compound
including, but not limited to, nucleic acids, peptides, polypeptides,
small molecules, and mixtures thereof. Non-limiting examples of nucleic
acids include interfering RNA molecules (e.g., siRNA, aiRNA, miRNA),
antisense oligonucleotides, plasmids, ribozymes, immunostimulatory
oligonucleotides, and mixtures thereof. Examples of peptides or
polypeptides include, without limitation, antibodies (e.g., polyclonal
antibodies, monoclonal antibodies, antibody fragments; humanized
antibodies, recombinant antibodies, recombinant human antibodies,
Primatized® antibodies), cytokines, growth factors, apoptotic factors,
differentiation-inducing factors, cell-surface receptors and their
ligands, hormones, and mixtures thereof. Examples of small molecules
include, but are not limited to, small organic molecules or compounds
such as any conventional agent or drug known to those of skill in the
art.

[0162] In some embodiments, the active agent is a therapeutic agent, or a
salt or derivative thereof. Therapeutic agent derivatives may be
therapeutically active themselves or they may be prodrugs, which become
active upon further modification. Thus, in one embodiment, a therapeutic
agent derivative retains some or all of the therapeutic activity as
compared to the unmodified agent, while in another embodiment, a
therapeutic agent derivative is a prodrug that lacks therapeutic
activity, but becomes active upon further modification.

[0163] A. Nucleic Acids

[0164] In certain embodiments, lipid particles of the present invention
are associated with a nucleic acid, resulting in a nucleic acid-lipid
particle (e.g., SNALP). In some embodiments, the nucleic acid is fully
encapsulated in the lipid particle. As used herein, the term "nucleic
acid" includes any oligonucleotide or polynucleotide, with fragments
containing up to 60 nucleotides generally termed oligonucleotides, and
longer fragments termed polynucleotides. In particular embodiments,
oligonucletoides of the invention are from about 15 to about 60
nucleotides in length. Nucleic acid may be administered alone in the
lipid particles of the invention, or in combination (e.g.,
co-administered) with lipid particles of the invention comprising
peptides, polypeptides, or small molecules such as conventional drugs.

[0165] In the context of this invention, the terms "polynucleotide" and
"oligonucleotide" refer to a polymer or oligomer of nucleotide or
nucleoside monomers consisting of naturally-occurring bases, sugars and
intersugar (backbone) linkages. The terms "polynucleotide" and
"oligonucleotide" also include polymers or oligomers comprising
non-naturally occurring monomers, or portions thereof, which function
similarly. Such modified or substituted oligonucleotides are often
preferred over native forms because of properties such as, for example,
enhanced cellular uptake, reduced immunogenicity, and increased stability
in the presence of nucleases.

[0166] Oligonucleotides are generally classified as
deoxyribooligonucleotides or ribooligonucleotides. A
deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose
joined covalently to phosphate at the 5' and 3' carbons of this sugar to
form an alternating, unbranched polymer. A ribooligonucleotide consists
of a similar repeating structure where the 5-carbon sugar is ribose.

[0167] The nucleic acid that is present in a lipid-nucleic acid particle
according to this invention includes any form of nucleic acid that is
known. The nucleic acids used herein can be single-stranded DNA or RNA,
or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of
double-stranded DNA are described herein and include, e.g., structural
genes, genes including control and termination regions, and
self-replicating systems such as viral or plasmid DNA. Examples of
double-stranded RNA are described herein and include, e.g., siRNA and
other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic
acids include, e.g., antisense oligonucleotides, ribozymes, mature miRNA,
and triplex-forming oligonucleotides.

[0168] Nucleic acids of the invention may be of various lengths, generally
dependent upon the particular form of nucleic acid. For example, in
particular embodiments, plasmids or genes may be from about 1,000 to
about 100,000 nucleotide residues in length. In particular embodiments,
oligonucleotides may range from about 10 to about 100 nucleotides in
length. In various related embodiments, oligonucleotides, both
single-stranded, double-stranded, and triple-stranded, may range in
length from about 10 to about 60 nucleotides, from about 15 to about 60
nucleotides, from about 20 to about 50 nucleotides, from about 15 to
about 30 nucleotides, or from about 20 to about 30 nucleotides in length.

[0169] In particular embodiments, an oligonucleotide (or a strand thereof)
of the invention specifically hybridizes to or is complementary to a
target polynucleotide sequence. The terms "specifically hybridizable" and
"complementary" as used herein indicate a sufficient degree of
complementarity such that stable and specific binding occurs between the
DNA or RNA target and the oligonucleotide. It is understood that an
oligonucleotide need not be 100% complementary to its target nucleic acid
sequence to be specifically hybridizable. In preferred embodiments, an
oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the target sequence interferes with the normal
function of the target sequence to cause a loss of utility or expression
therefrom, and there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide to non-target sequences under
conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or therapeutic
treatment, or, in the case of in vitro assays, under conditions in which
the assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,
or more base substitutions as compared to the region of a gene or mRNA
sequence that it is targeting or to which it specifically hybridizes.

[0170] 1. siRNA

[0171] The siRNA component of the nucleic acid-lipid particles of the
present invention is capable of silencing the expression of a target gene
of interest. Each strand of the siRNA duplex is typically about 15 to
about 60 nucleotides in length, preferably about 15 to about 30
nucleotides in length. In certain embodiments, the siRNA comprises at
least one modified nucleotide. The modified siRNA is generally less
immunostimulatory than a corresponding unmodified siRNA sequence and
retains RNAi activity against the target gene of interest. In some
embodiments, the modified siRNA contains at least one 2'OMe purine or
pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uridine,
2'OMe-adenosine, and/or 2'OMe-cytosine nucleotide. In preferred
embodiments, one or more of the uridine and/or guanosine nucleotides are
modified. The modified nucleotides can be present in one strand (i.e.,
sense or antisense) or both strands of the siRNA. The siRNA sequences may
have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al.,
Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)), or
may lack overhangs (i.e., have blunt ends).

[0177] Suitable siRNA sequences can be identified using any means known in
the art. Typically, the methods described in Elbashir et al., Nature,
411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) are
combined with rational design rules set forth in Reynolds et al., Nature
Biotech., 22(3):326-330 (2004).

[0178] Generally, the nucleotide sequence 3' of the AUG start codon of a
transcript from the target gene of interest is scanned for dinucleotide
sequences (e.g., AA, NA, CC, GG, or UU, wherein N═C, G, or U) (see,
e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotides
immediately 3' to the dinucleotide sequences are identified as potential
siRNA sequences (i.e., a target sequence or a sense strand sequence).
Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides
immediately 3' to the dinucleotide sequences are identified as potential
siRNA sequences. In some embodiments, the dinucleotide sequence is an AA
or NA sequence and the 19 nucleotides immediately 3' to the AA or NA
dinucleotide are identified as potential siRNA sequences. siRNA sequences
are usually spaced at different positions along the length of the target
gene. To further enhance silencing efficiency of the siRNA sequences,
potential siRNA sequences may be analyzed to identify sites that do not
contain regions of homology to other coding sequences, e.g., in the
target cell or organism. For example, a suitable siRNA sequence of about
21 base pairs typically will not have more than 16-17 contiguous base
pairs of homology to coding sequences in the target cell or organism. If
the siRNA sequences are to be expressed from an RNA Pol III promoter,
siRNA sequences lacking more than 4 contiguous A's or T's are selected.

[0179] Once a potential siRNA sequence has been identified, a
complementary sequence (i.e., an antisense strand sequence) can be
designed. A potential siRNA sequence can also be analyzed using a variety
of criteria known in the art. For example, to enhance their silencing
efficiency, the siRNA sequences may be analyzed by a rational design
algorithm to identify sequences that have one or more of the following
features: (1) G/C content of about 25% to about 60% G/C; (2) at least 3
A/Us at positions 15-19 of the sense strand; (3) no internal repeats; (4)
an A at position 19 of the sense strand; (5) an A at position 3 of the
sense strand; (6) a U at position 10 of the sense strand; (7) no G/C at
position 19 of the sense strand; and (8) no G at position 13 of the sense
strand. siRNA design tools that incorporate algorithms that assign
suitable values of each of these features and are useful for selection of
siRNA can be found at, e.g., http://boz094.ust.hk/RNAi/siRNA. One of
skill in the art will appreciate that sequences with one or more of the
foregoing characteristics may be selected for further analysis and
testing as potential siRNA sequences.

[0180] Additionally, potential siRNA sequences with one or more of the
following criteria can often be eliminated as siRNA: (1) sequences
comprising a stretch of 4 or more of the same base in a row; (2)
sequences comprising homopolymers of Gs (i.e., to reduce possible
non-specific effects due to structural characteristics of these polymers;
(3) sequences comprising triple base motifs (e.g., GGG, CCC, AAA, or
TTT); (4) sequences comprising stretches of 7 or more G/Cs in a row; and
(5) sequences comprising direct repeats of 4 or more bases within the
candidates resulting in internal fold-back structures. However, one of
skill in the art will appreciate that sequences with one or more of the
foregoing characteristics may still be selected for further analysis and
testing as potential siRNA sequences.

[0181] In some embodiments, potential siRNA sequences may be further
analyzed based on siRNA duplex asymmetry as described in, e.g., Khvorova
et al., Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208
(2003). In other embodiments, potential siRNA sequences may be further
analyzed based on secondary structure at the target site as described in,
e.g., Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,
secondary structure at the target site can be modeled using the Mfold
algorithm (available at
http://www.bioinfo.rpi.edu/applications/mfold/rna/forml.cgi) to select
siRNA sequences which favor accessibility at the target site where less
secondary structure in the form of base-pairing and stem-loops is
present.

[0182] Once a potential siRNA sequence has been identified, the sequence
can be analyzed for the presence of any immunostimulatory properties,
e.g., using an in vitro cytokine assay or an in vivo animal model. Motifs
in the sense and/or antisense strand of the siRNA sequence such as
GU-rich motifs (e.g., 5'-GU-3',5'-UGU-3',5'-GUGU-3',5'-UGUGU-3', etc.)
can also provide an indication of whether the sequence may be
immunostimulatory. Once an siRNA molecule is found to be
immunostimulatory, it can then be modified to decrease its
immunostimulatory properties as described herein. As a non-limiting
example, an siRNA sequence can be contacted with a mammalian responder
cell under conditions such that the cell produces a detectable immune
response to determine whether the siRNA is an immunostimulatory or a
non-immunostimulatory siRNA. The mammalian responder cell may be from a
naive mammal (i.e., a mammal that has not previously been in contact with
the gene product of the siRNA sequence). The mammalian responder cell may
be, e.g., a peripheral blood mononuclear cell (PBMC), a macrophage, and
the like. The detectable immune response may comprise production of a
cytokine or growth factor such as, e.g., TNF-α, IFN-α,
IFN-β, IFN-γ, IL-6, IL-12, or a combination thereof. An siRNA
molecule identified as being immunostimulatory can then be modified to
decrease its immunostimulatory properties by replacing at least one of
the nucleotides on the sense and/or antisense strand with modified
nucleotides. For example, less than about 30% (e.g., less than about 30%,
25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-stranded
region of the siRNA duplex can be replaced with modified nucleotides such
as 2'OMe nucleotides. The modified siRNA can then be contacted with a
mammalian responder cell as described above to confirm that its
immunostimulatory properties have been reduced or abrogated.

[0184] A non-limiting example of an in vivo model for detecting an immune
response includes an in vivo mouse cytokine induction assay as described
in, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certain
embodiments, the assay that can be performed as follows: (1) siRNA can be
administered by standard intravenous injection in the lateral tail vein;
(2) blood can be collected by cardiac puncture about 6 hours after
administration and processed as plasma for cytokine analysis; and (3)
cytokines can be quantified using sandwich ELISA kits according to the
manufacturer's instructions (e.g., mouse and human IFN-α (PBL
Biomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience;
San Diego, Calif.); and mouse IL-6, TNF-α, and IFN-γ (BD
Biosciences; San Diego, Calif.)).

[0185] Monoclonal antibodies that specifically bind cytokines and growth
factors are commercially available from multiple sources and can be
generated using methods known in the art (see, e.g., Kohler et al.,
Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY
MANUAL, Cold Spring Harbor Publication, New York (1999)). Generation of
monoclonal antibodies has been previously described and can be
accomplished by any means known in the art (Buhring et al., in Hybridoma,
Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, the monoclonal
antibody is labeled (e.g., with any composition detectable by
spectroscopic, photochemical, biochemical, electrical, optical, or
chemical means) to facilitate detection.

[0186] b. Generating siRNA Molecules

[0187] siRNA can be provided in several forms including, e.g., as one or
more isolated small-interfering RNA (siRNA) duplexes, as longer
double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a
transcriptional cassette in a DNA plasmid. The siRNA sequences may have
overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al.,
Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001), or may
lack overhangs (i.e., to have blunt ends).

[0188] An RNA population can be used to provide long precursor RNAs, or
long precursor RNAs that have substantial or complete identity to a
selected target sequence can be used to make the siRNA. The RNAs can be
isolated from cells or tissue, synthesized, and/or cloned according to
methods well known to those of skill in the art. The RNA can be a mixed
population (obtained from cells or tissue, transcribed from cDNA,
subtracted, selected, etc.), or can represent a single target sequence.
RNA can be naturally occurring (e.g., isolated from tissue or cell
samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR
products or a cloned cDNA), or chemically synthesized.

[0189] To form a long dsRNA, for synthetic RNAs, the complement is also
transcribed in vitro and hybridized to form a dsRNA. If a naturally
occurring RNA population is used, the RNA complements are also provided
(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,
by transcribing cDNAs corresponding to the RNA population, or by using
RNA polymerases. The precursor RNAs are then hybridized to form double
stranded RNAs for digestion. The dsRNAs can be directly administered to a
subject or can be digested in vitro prior to administration.

[0190] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic
acids, making and screening cDNA libraries, and performing PCR are well
known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);
Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,
U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods
and Applications (Innis et al., eds, 1990)). Expression libraries are
also well known to those of skill in the art. Additional basic texts
disclosing the general methods of use in this invention include Sambrook
et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler,
Gene Transfer and Expression: A Laboratory Manual (1990); and Current
Protocols in Molecular Biology (Ausubel et al., eds., 1994). The
disclosures of these references are herein incorporated by reference in
their entirety for all purposes.

[0191] Preferably, siRNA are chemically synthesized. The oligonucleotides
that comprise the siRNA molecules of the invention can be synthesized
using any of a variety of techniques known in the art, such as those
described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe
et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids
Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59
(1997). The synthesis of oligonucleotides makes use of common nucleic
acid protecting and coupling groups, such as dimethoxytrityl at the
5'-end and phosphoramidites at the 3'-end. As a non-limiting example,
small scale syntheses can be conducted on an Applied Biosystems
synthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses
at the 0.2 μmol scale can be performed on a 96-well plate synthesizer
from Protogene (Palo Alto, Calif.). However, a larger or smaller scale of
synthesis is also within the scope of this invention. Suitable reagents
for oligonucleotide synthesis, methods for RNA deprotection, and methods
for RNA purification are known to those of skill in the art.

[0192] siRNA molecules can also be synthesized via a tandem synthesis
technique, wherein both strands are synthesized as a single continuous
oligonucleotide fragment or strand separated by a cleavable linker that
is subsequently cleaved to provide separate fragments or strands that
hybridize to form the siRNA duplex. The linker can be a polynucleotide
linker or a non-nucleotide linker. The tandem synthesis of siRNA can be
readily adapted to both multiwell/multiplate synthesis platforms as well
as large scale synthesis platforms employing batch reactors, synthesis
columns, and the like. Alternatively, siRNA molecules can be assembled
from two distinct oligonucleotides, wherein one oligonucleotide comprises
the sense strand and the other comprises the antisense strand of the
siRNA. For example, each strand can be synthesized separately and joined
together by hybridization or ligation following synthesis and/or
deprotection. In certain other instances, siRNA molecules can be
synthesized as a single continuous oligonucleotide fragment, where the
self-complementary sense and antisense regions hybridize to form an siRNA
duplex having hairpin secondary structure.

[0193] c. Modifying siRNA Sequences

[0194] In certain aspects, siRNA molecules comprise a duplex having two
strands and at least one modified nucleotide in the double-stranded
region, wherein each strand is about 15 to about 60 nucleotides in
length. Advantageously, the modified siRNA is less immunostimulatory than
a corresponding unmodified siRNA sequence, but retains the capability of
silencing the expression of a target sequence. In preferred embodiments,
the degree of chemical modifications introduced into the siRNA molecule
strikes a balance between reduction or abrogation of the
immunostimulatory properties of the siRNA and retention of RNAi activity.
As a non-limiting example, an siRNA molecule that targets a gene of
interest can be minimally modified (e.g., less than about 30%, 25%, 20%,
15%, 10%, or 5% modified) at selective uridine and/or guanosine
nucleotides within the siRNA duplex to eliminate the immune response
generated by the siRNA while retaining its capability to silence target
gene expression.

[0195] Examples of modified nucleotides suitable for use in the invention
include, but are not limited to, ribonucleotides having a 2'-O-methyl
(2'OMe), 2'-deoxy-2'-fluoro(2'F), 2'-deoxy, 5-C-methyl,
2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group.
Modified nucleotides having a Northern conformation such as those
described in, e.g., Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag Ed. (1984), are also suitable for use in siRNA molecules.
Such modified nucleotides include, without limitation, locked nucleic
acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl)
nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides,
2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoro(2'F) nucleotides,
2'-deoxy-2'-chloro(2'Cl) nucleotides, and 2'-azido nucleotides. In
certain instances, the siRNA molecules described herein include one or
more G-clamp nucleotides. A G-clamp nucleotide refers to a modified
cytosine analog wherein the modifications confer the ability to hydrogen
bond both Watson-Crick and Hoogsteen faces of a complementary guanine
nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. Soc.,
120:8531-8532 (1998)). In addition, nucleotides having a nucleotide base
analog such as, for example, C-phenyl, C-naphthyl, other aromatic
derivatives, inosine, azole carboxamides, and nitroazole derivatives such
as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see,
e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated
into siRNA molecules.

[0197] In some embodiments, the sense and/or antisense strand of the siRNA
molecule can further comprise a 3'-terminal overhang having about 1 to
about 4 (e.g., 1, 2, 3, or 4) 2'-deoxy ribonucleotides and/or any
combination of modified and unmodified nucleotides. Additional examples
of modified nucleotides and types of chemical modifications that can be
introduced into siRNA molecules are described, e.g., in UK Patent No. GB
2,397,818 B and U.S. Patent Publication Nos. 20040192626, 20050282188,
and 20070135372, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.

[0198] The siRNA molecules described herein can optionally comprise one or
more non-nucleotides in one or both strands of the siRNA. As used herein,
the term "non-nucleotide" refers to any group or compound that can be
incorporated into a nucleic acid chain in the place of one or more
nucleotide units, including sugar and/or phosphate substitutions, and
allows the remaining bases to exhibit their activity. The group or
compound is abasic in that it does not contain a commonly recognized
nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine
and therefore lacks a base at the 1'-position.

[0199] In other embodiments, chemical modification of the siRNA comprises
attaching a conjugate to the siRNA molecule. The conjugate can be
attached at the 5' and/or 3'-end of the sense and/or antisense strand of
the siRNA via a covalent attachment such as, e.g., a biodegradable
linker. The conjugate can also be attached to the siRNA, e.g., through a
carbamate group or other linking group (see, e.g., U.S. Patent
Publication Nos. 20050074771, 20050043219, and 20050158727). In certain
instances, the conjugate is a molecule that facilitates the delivery of
the siRNA into a cell. Examples of conjugate molecules suitable for
attachment to siRNA include, without limitation, steroids such as
cholesterol, glycols such as polyethylene glycol (PEG), human serum
albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates
(e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g.,
galactose, galactosamine, N-acetyl galactosamine, glucose, mannose,
fructose, fucose, etc.), phospholipids, peptides, ligands for cellular
receptors capable of mediating cellular uptake, and combinations thereof
(see, e.g., U.S. Patent Publication Nos. 20030130186, 20040110296, and
20040249178; U.S. Pat. No. 6,753,423). Other examples include the
lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid,
small molecule, oligosaccharide, carbohydrate cluster, intercalator,
minor groove binder, cleaving agent, and cross-linking agent conjugate
molecules described in U.S. Patent Publication Nos. 20050119470 and
20050107325. Yet other examples include the 2'-O-alkyl amine,
2'-O-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine,
cationic peptide, guanidinium group, amidininium group, cationic amino
acid conjugate molecules described in U.S. Patent Publication No.
20050153337. Additional examples include the hydrophobic group, membrane
active compound, cell penetrating compound, cell targeting signal,
interaction modifier, and steric stabilizer conjugate molecules described
in U.S. Patent Publication No. 20040167090. Further examples include the
conjugate molecules described in U.S. Patent Publication No. 20050239739.
The type of conjugate used and the extent of conjugation to the siRNA
molecule can be evaluated for improved pharmacokinetic profiles,
bioavailability, and/or stability of the siRNA while retaining RNAi
activity. As such, one skilled in the art can screen siRNA molecules
having various conjugates attached thereto to identify ones having
improved properties and full RNAi activity using any of a variety of
well-known in vitro cell culture or in vivo animal models. The
disclosures of the above-described patent documents are herein
incorporated by reference in their entirety for all purposes.

[0200] d. Target Genes

[0201] The siRNA component of the nucleic acid-lipid particles described
herein can be used to downregulate or silence the translation (i.e.,
expression) of a gene of interest. Genes of interest include, but are not
limited to, genes associated with viral infection and survival, genes
associated with metabolic diseases and disorders (e.g., liver diseases
and disorders), genes associated with tumorigenesis and cell
transformation (e.g., cancer), angiogenic genes, immunomodulator genes
such as those associated with inflammatory and autoimmune responses,
ligand receptor genes, and genes associated with neurodegenerative
disorders.

[0209] Silencing of sequences that encode DNA repair enzymes find use in
combination with the administration of chemotherapeutic agents (Collis et
al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associated
with tumor migration are also target sequences of interest, for example,
integrins, selectins, and metalloproteinases. The foregoing examples are
not exclusive. Those of skill in the art will understand that any whole
or partial gene sequence that facilitates or promotes tumorigenesis or
cell transformation, tumor growth, or tumor migration can be included as
a template sequence.

[0210] Angiogenic genes are able to promote the formation of new vessels.
Of particular interest is vascular endothelial growth factor (VEGF)
(Reich et al., Mol. Vis., 9:210 (2003)) or VEGFR. siRNA sequences that
target VEGFR are set forth in, e.g., GB 2396864; U.S. Patent Publication
No. 20040142895; and CA 2456444, the disclosures of which are herein
incorporated by reference in their entirety for all purposes.

[0211] Anti-angiogenic genes are able to inhibit neovascularization. These
genes are particularly useful for treating those cancers in which
angiogenesis plays a role in the pathological development of the disease.
Examples of anti-angiogenic genes include, but are not limited to,
endostatin (see, e.g., U.S. Pat. No. 6,174,861), angiostatin (see, e.g.,
U.S. Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J.
Pathol., 188: 369-377 (1999)), the disclosures of which are herein
incorporated by reference in their entirety for all purposes.

[0214] In addition to its utility in silencing the expression of any of
the above-described genes for therapeutic purposes, the siRNA described
herein are also useful in research and development applications as well
as diagnostic, prophylactic, prognostic, clinical, and other healthcare
applications. As a non-limiting example, the siRNA can be used in target
validation studies directed at testing whether a gene of interest has the
potential to be a therapeutic target. The siRNA can also be used in
target identification studies aimed at discovering genes as potential
therapeutic targets.

[0215] 2. aiRNA

[0216] Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit the
RNA-induced silencing complex (RISC) and lead to effective silencing of a
variety of genes in mammalian cells by mediating sequence-specific
cleavage of the target sequence between nucleotide 10 and 11 relative to
the 5' end of the antisense strand (Sun et al., Nat. Biotech.,
26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNA
duplex having a sense strand and an antisense strand, wherein the duplex
contains overhangs at the 3' and 5' ends of the antisense strand. The
aiRNA is generally asymmetric because the sense strand is shorter on both
ends when compared to the complementary antisense strand. In some
aspects, aiRNA molecules may be designed, synthesized, and annealed under
conditions similar to those used for siRNA molecules. As a non-limiting
example, aiRNA sequences may be selected and generated using the methods
described above for selecting siRNA sequences.

[0217] In another embodiment, aiRNA duplexes of various lengths (e.g.,
about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more
typically 12, 13, 14, 15, 16, 17, 18, 19, or base pairs) may be designed
with overhangs at the 3' and 5' ends of the antisense strand to target an
mRNA of interest. In certain instances, the sense strand of the aiRNA
molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides
in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20
nucleotides in length. In certain other instances, the antisense strand
of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in
length, more typically about 15-30, 15-25, or 19-25 nucleotides in
length, and is preferably about 20-24, 21-22, or 21-23 nucleotides in
length.

[0218] In some embodiments, the 5' antisense overhang contains one, two,
three, four, or more nontargeting nucleotides (e.g., "AA", "UU", "dTdT",
etc.). In other embodiments, the 3' antisense overhang contains one, two,
three, four, or more nontargeting nucleotides (e.g., "AA", "UU", "dTdT",
etc.). In certain aspects, the aiRNA molecules described herein may
comprise one or more modified nucleotides, e.g., in the double-stranded
(duplex) region and/or in the antisense overhangs. As a non-limiting
example, aiRNA sequences may comprise one or more of the modified
nucleotides described above for siRNA sequences. In a preferred
embodiment, the aiRNA molecule comprises 2'OMe nucleotides such as, for
example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or
mixtures thereof.

[0219] In certain embodiments, aiRNA molecules may comprise an antisense
strand which corresponds to the antisense strand of an siRNA molecule,
e.g., one of the siRNA molecules described herein. In other embodiments,
aiRNA molecules may be used to silence the expression of any of the
target genes set forth above, such as, e.g., genes associated with viral
infection and survival, genes associated with metabolic diseases and
disorders, genes associated with tumorigenesis and cell transformation,
angiogenic genes, immunomodulator genes such as those associated with
inflammatory and autoimmune responses, ligand receptor genes, and genes
associated with neurodegenerative disorders.

[0220] 3. miRNA

[0221] Generally, microRNAs (miRNA) are single-stranded RNA molecules of
about 21-23 nucleotides in length which regulate gene expression. miRNAs
are encoded by genes from whose DNA they are transcribed, but miRNAs are
not translated into protein (non-coding RNA); instead, each primary
transcript (a pri-miRNA) is processed into a short stem-loop structure
called a pre-miRNA and finally into a functional mature miRNA. Mature
miRNA molecules are either partially or completely complementary to one
or more messenger RNA (mRNA) molecules, and their main function is to
downregulate gene expression. The identification of miRNA molecules is
described, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau et
al., Science, 294:858-862; and Lee et al., Science, 294:862-864.

[0222] The genes encoding miRNA are much longer than the processed mature
miRNA molecule. miRNA are first transcribed as primary transcripts or
pri-miRNA with a cap and poly-A tail and processed to short,
˜70-nucleotide stem-loop structures known as pre-miRNA in the cell
nucleus. This processing is performed in animals by a protein complex
known as the Microprocessor complex, consisting of the nuclease Drosha
and the double-stranded RNA binding protein Pasha (Denli et al., Nature,
432:231-235 (2004)). These pre-miRNA are then processed to mature miRNA
in the cytoplasm by interaction with the endonuclease Dicer, which also
initiates the formation of the RNA-induced silencing complex (RISC)
(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strand or
antisense strand of DNA can function as templates to give rise to miRNA.

[0223] When Dicer cleaves the pre-miRNA stem-loop, two complementary short
RNA molecules are formed, but only one is integrated into the RISC
complex. This strand is known as the guide strand and is selected by the
argonaute protein, the catalytically active RNase in the RISC complex, on
the basis of the stability of the 5' end (Preall et al., Curr. Biol.,
16:530-535 (2006)). The remaining strand, known as the anti-guide or
passenger strand, is degraded as a RISC complex substrate (Gregory et
al., Cell, 123:631-640 (2005)). After integration into the active RISC
complex, miRNAs base pair with their complementary mRNA molecules and
induce target mRNA degradation and/or translational silencing.

[0224] Mammalian miRNA molecules are usually complementary to a site in
the 3' UTR of the target mRNA sequence. In certain instances, the
annealing of the miRNA to the target mRNA inhibits protein translation by
blocking the protein translation machinery. In certain other instances,
the annealing of the miRNA to the target mRNA facilitates the cleavage
and degradation of the target mRNA through a process similar to RNA
interference (RNAi). miRNA may also target methylation of genomic sites
which correspond to targeted mRNA. Generally, miRNA function in
association with a complement of proteins collectively termed the miRNP.

[0225] In certain aspects, the miRNA molecules described herein are about
15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in
length, more typically about 15-30, 15-25, or 19-25 nucleotides in
length, and are preferably about 20-24, 21-22, or 21-23 nucleotides in
length. In certain other aspects, miRNA molecules may comprise one or
more modified nucleotides. As a non-limiting example, miRNA sequences may
comprise one or more of the modified nucleotides described above for
siRNA sequences. In a preferred embodiment, the miRNA molecule comprises
2'OMe nucleotides such as, for example, 2'OMe-guanosine nucleotides,
2'OMe-uridine nucleotides, or mixtures thereof.

[0226] In some embodiments, miRNA molecules may be used to silence the
expression of any of the target genes set forth above, such as, e.g.,
genes associated with viral infection and survival, genes associated with
metabolic diseases and disorders, genes associated with tumorigenesis and
cell transformation, angiogenic genes, immunomodulator genes such as
those associated with inflammatory and autoimmune responses, ligand
receptor genes, and genes associated with neurodegenerative disorders.

[0227] In other embodiments, one or more agents that block the activity of
a miRNA targeting an mRNA of interest are administered using a lipid
particle of the invention (e.g., a nucleic acid-lipid particle). Examples
of blocking agents include, but are not limited to, steric blocking
oligonucleotides, locked nucleic acid oligonucleotides, and Morpholino
oligonucleotides. Such blocking agents may bind directly to the miRNA or
to the miRNA binding site on the target mRNA.

[0228] 4. Antisense Oligonucleotides

[0229] In one embodiment, the nucleic acid is an antisense oligonucleotide
directed to a target gene or sequence of interest. The terms "antisense
oligonucleotide" or "antisense" include oligonucleotides that are
complementary to a targeted polynucleotide sequence. Antisense
oligonucleotides are single strands of DNA or RNA that are complementary
to a chosen sequence. Antisense RNA oligonucleotides prevent the
translation of complementary RNA strands by binding to the RNA. Antisense
DNA oligonucleotides can be used to target a specific, complementary
(coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be
degraded by the enzyme RNase H. In a particular embodiment, antisense
oligonucleotides comprise from about 10 to about 60 nucleotides, more
preferably from about 15 to about 30 nucleotides. The term also
encompasses antisense oligonucleotides that may not be exactly
complementary to the desired target gene. Thus, the invention can be
utilized in instances where non-target specific-activities are found with
antisense, or where an antisense sequence containing one or more
mismatches with the target sequence is the most preferred for a
particular use.

[0230] Antisense oligonucleotides have been demonstrated to be effective
and targeted inhibitors of protein synthesis, and, consequently, can be
used to specifically inhibit protein synthesis by a targeted gene. The
efficacy of antisense oligonucleotides for inhibiting protein synthesis
is well established. For example, the synthesis of polygalactauronase and
the muscarine type 2 acetylcholine receptor are inhibited by antisense
oligonucleotides directed to their respective mRNA sequences (see, U.S.
Pat. Nos. 5,739,119 and 5,759,829). Furthermore, examples of antisense
inhibition have been demonstrated with the nuclear protein cyclin, the
multiple drug resistance gene (MDR1), ICAM-1, E-selectin, STK-1, striatal
GABAA receptor, and human EGF (see, Jaskulski et al., Science, 240:1544-6
(1988); Vasanthakumar et al., Cancer Commun., 1:225-32 (1989); Penis et
al., Brain Res Mol Brain Res., 15; 57:310-20 (1998); and U.S. Pat. Nos.
5,801,154; 5,789,573; 5,718,709 and 5,610,288). Moreover, antisense
constructs have also been described that inhibit and can be used to treat
a variety of abnormal cellular proliferations, e.g., cancer (see, U.S.
Pat. Nos. 5,747,470; 5,591,317; and 5,783,683). The disclosures of these
references are herein incorporated by reference in their entirety for all
purposes.

[0231] Methods of producing antisense oligonucleotides are known in the
art and can be readily adapted to produce an antisense oligonucleotide
that targets any polynucleotide sequence. Selection of antisense
oligonucleotide sequences specific for a given target sequence is based
upon analysis of the chosen target sequence and determination of
secondary structure, Tm, binding energy, and relative stability.
Antisense oligonucleotides may be selected based upon their relative
inability to form dimers, hairpins, or other secondary structures that
would reduce or prohibit specific binding to the target mRNA in a host
cell. Highly preferred target regions of the mRNA include those regions
at or near the AUG translation initiation codon and those sequences that
are substantially complementary to 5' regions of the mRNA. These
secondary structure analyses and target site selection considerations can
be performed, for example, using v.4 of the OLIGO primer analysis
software (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithm
software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0232] 5. Ribozymes

[0233] According to another embodiment of the invention, nucleic
acid-lipid particles are associated with ribozymes. Ribozymes are
RNA-protein complexes having specific catalytic domains that possess
endonuclease activity (see, Kim et al., Proc. Natl. Acad. Sci. USA.,
84:8788-92 (1987); and Forster et a, Cell, 49:211-20 (1987)). For
example, a large number of ribozymes accelerate phosphoester transfer
reactions with a high degree of specificity, often cleaving only one of
several phosphoesters in an oligonucleotide substrate (see, Cech et al.,
Cell, 27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990);
Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity has
been attributed to the requirement that the substrate bind via specific
base-pairing interactions to the internal guide sequence ("IGS") of the
ribozyme prior to chemical reaction.

[0234] At least six basic varieties of naturally-occurring enzymatic RNA
molecules are known presently. Each can catalyze the hydrolysis of RNA
phosphodiester bonds in trans (and thus can cleave other RNA molecules)
under physiological conditions. In general, enzymatic nucleic acids act
by first binding to a target RNA. Such binding occurs through the target
binding portion of an enzymatic nucleic acid which is held in close
proximity to an enzymatic portion of the molecule that acts to cleave the
target RNA. Thus, the enzymatic nucleic acid first recognizes and then
binds a target RNA through complementary base-pairing, and once bound to
the correct site, acts enzymatically to cut the target RNA. Strategic
cleavage of such a target RNA will destroy its ability to direct
synthesis of an encoded protein. After an enzymatic nucleic acid has
bound and cleaved its RNA target, it is released from that RNA to search
for another target and can repeatedly bind and cleave new targets.

[0235] The enzymatic nucleic acid molecule may be formed in a hammerhead,
hairpin, hepatitis 8 virus, group I intron or RNaseP RNA (in association
with an RNA guide sequence), or Neurospora VS RNA motif, for example.
Specific examples of hammerhead motifs are described in, e.g., Rossi et
al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs
are described in, e.g., EP 0360257, Hampel et al., Biochemistry,
28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990);
and U.S. Pat. No. 5,631,359. An example of the hepatitis 8 virus motif is
described in, e.g., Perrotta et al., Biochemistry, 31:11843-52 (1992). An
example of the RNaseP motif is described in, e.g., Guerrier-Takada et
al., Cell, 35:849-57 (1983). Examples of the Neurospora VS RNA ribozyme
motif is described in, e.g., Saville et al., Cell, 61:685-96 (1990);
Saville et al., Proc. Natl. Acad. Sci. USA, 88:8826-30 (1991); Collins et
al., Biochemistry, 32:2795-9 (1993). An example of the Group I intron is
described in, e.g., U.S. Pat. No. 4,987,071. Important characteristics of
enzymatic nucleic acid molecules used according to the invention are that
they have a specific substrate binding site which is complementary to one
or more of the target gene DNA or RNA regions, and that they have
nucleotide sequences within or surrounding that substrate binding site
which impart an RNA cleaving activity to the molecule. Thus, the ribozyme
constructs need not be limited to specific motifs mentioned herein. The
disclosures of these references are herein incorporated by reference in
their entirety for all purposes.

[0236] Methods of producing a ribozyme targeted to any polynucleotide
sequence are known in the art. Ribozymes may be designed as described in,
e.g., PCT Publication Nos. WO 93/23569 and WO 94/02595, and synthesized
to be tested in vitro and/or in vivo as described therein. The
disclosures of these PCT publications are herein incorporated by
reference in their entirety for all purposes.

[0237] Ribozyme activity can be optimized by altering the length of the
ribozyme binding arms or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases (see,
e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO 91/03162, and WO
94/13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, which describe
various chemical modifications that can be made to the sugar moieties of
enzymatic RNA molecules, the disclosures of which are each herein
incorporated by reference in their entirety for all purposes),
modifications which enhance their efficacy in cells, and removal of stem
II bases to shorten RNA synthesis times and reduce chemical requirements.

[0238] 6. Immunostimulatory Oligonucleotides

[0239] Nucleic acids associated with lipid paticles of the present
invention may be immunostimulatory, including immunostimulatory
oligonucleotides (ISS; single- or double-stranded) capable of inducing an
immune response when administered to a subject, which may be a mammal
such as a human. ISS include, e.g., certain palindromes leading to
hairpin secondary structures (see, Yamamoto et al., J. Immunol.,
148:4072-6 (1992)), or CpG motifs, as well as other known ISS features
(such as multi-G domains; see; PCT Publication No. WO 96/11266, the
disclosure of which is herein incorporated by reference in its entirety
for all purposes).

[0240] Immunostimulatory nucleic acids are considered to be non-sequence
specific when it is not required that they specifically bind to and
reduce the expression of a target sequence in order to provoke an immune
response. Thus, certain immunostimulatory nucleic acids may comprise a
sequence corresponding to a region of a naturally-occurring gene or mRNA,
but they may still be considered non-sequence specific immunostimulatory
nucleic acids.

[0241] In one embodiment, the immunostimulatory nucleic acid or
oligonucleotide comprises at least one CpG dinucleotide. The
oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In
another embodiment, the immunostimulatory nucleic acid comprises at least
one CpG dinucleotide having a methylated cytosine. In one embodiment, the
nucleic acid comprises a single CpG dinucleotide, wherein the cytosine in
the CpG dinucleotide is methylated. In an alternative embodiment, the
nucleic acid comprises at least two CpG dinucleotides, wherein at least
one cytosine in the CpG dinucleotides is methylated. In a further
embodiment, each cytosine in the CpG dinucleotides present in the
sequence is methylated. In another embodiment, the nucleic acid comprises
a plurality of CpG dinucleotides, wherein at least one of the CpG
dinucleotides comprises a methylated cytosine. Examples of
immunostimulatory oligonucleotides suitable for use in the compositions
and methods of the present invention are described in PCT Application No.
PCT/US08/88676, filed Dec. 31, 2008, PCT Publication Nos. WO 02/069369
and WO 01/15726, U.S. Pat. No. 6,406,705, and Raney et al., J. Pharm.
Exper. Ther., 298:1185-92 (2001), the disclosures of which are each
herein incorporated by reference in their entirety for all purposes. In
certain embodiments, the oligonucleotides used in the compositions and
methods of the invention have a phosphodiester ("PO") backbone or a
phosphorothioate ("PS") backbone, and/or at least one methylated cytosine
residue in a CpG motif.

[0242] B. Other Active Agents

[0243] In certain embodiments, the active agent associated with the lipid
paticles of the invention may comprise one or more therapeutic proteins,
polypeptides, or small organic molecules or compounds. Non-limiting
examples of such therapeutically effective agents or drugs include
oncology drugs (e.g., chemotherapy drugs, hormonal therapaeutic agents,
immunotherapeutic agents, radiotherapeutic agents, etc.), lipid-lowering
agents, anti-viral drugs, anti-inflammatory compounds, antidepressants,
stimulants, analgesics, antibiotics, birth control medication,
antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal
transduction inhibitors, cardiovascular drugs such as anti-arrhythmic
agents, hormones, vasoconstrictors, and steroids. These active agents may
be administered alone in the lipid particles of the invention, or in
combination (e.g., co-administered) with lipid particles of the invention
comprising nucleic acid such as interfering RNA.

[0251] The lipid particles of the invention typically comprise an active
agent or therapeutic agent, a cationic lipid, a non-cationic lipid, and a
conjugated lipid that inhibits aggregation of particles. In some
embodiments, the active agent or therapeutic agent is fully encapsulated
within the lipid portion of the lipid particle such that the active agent
or therapeutic agent in the lipid particle is resistant in aqueous
solution to enzymatic degradation, e.g., by a nuclease or protease. In
other embodiments, the lipid particles described herein are substantially
non-toxic to mammals such as humans. The lipid particles of the invention
typically have a mean diameter of from about 40 nm to about 150 nm, from
about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about
70 nm to about 110 nm, or from about 70 to about 90 nm.

[0252] In preferred embodiments, the lipid particles of the invention are
serum-stable nucleic acid-lipid particles (SNALP) which comprise an
interfering RNA (e.g., siRNA, aiRNA, and/or miRNA), a cationic lipid
(e.g., a cationic lipid of Formulas I, II, and/or III), a non-cationic
lipid (e.g., cholesterol alone or mixtures of one or more phospholipids
and cholesterol), and a conjugated lipid that inhibits aggregation of the
particles (e.g., one or more PEG-lipid conjugates). The SNALP may
comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified
and/or modified interfering RNA molecules. Nucleic acid-lipid particles
and their method of preparation are described in, e.g., U.S. Pat. Nos.
5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and
6,320,017; and PCT Publication No. WO 96/40964, the disclosures of which
are each herein incorporated by reference in their entirety for all
purposes.

[0253] A. Cationic Lipids

[0254] Any of a variety of cationic lipids may be used in the lipid
particles of the invention (e.g., SNALP), either alone or in combination
with one or more other cationic lipid species or non-cationic lipid
species.

[0255] Cationic lipids which are useful in the present invention can be
any of a number of lipid species which carry a net positive charge at
physiological pH. Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),
1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),
3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimet-
hyl-1-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl
spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1--
(cis,cis-9,12-octadecadienoxy)propane (CLinDMA),
2-[5'-(cholest-5-en-3β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',-
1-2'-octadecadienoxy)propane (CpLinDMA),
N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),
1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),
1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),
1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixtures
thereof A number of these lipids and related analogs have been described
in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat.
Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and
5,785,992; and PCT Publication No. WO 96/10390, the disclosures of which
are each herein incorporated by reference in their entirety for all
purposes. Additionally, a number of commercial preparations of cationic
lipids are available and can be used in the present invention. These
include, e.g., LIPOFECTIN® (commercially available cationic liposomes
comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y., USA);
LIPOFECTAMINE® (commercially available cationic liposomes comprising
DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM® (commercially
available cationic liposomes comprising DOGS from Promega Corp., Madison,
Wis., USA).

[0256] Additionally, cationic lipids of Formula I having the following
structures are useful in the present invention.

##STR00001##

wherein R1 and R2 are independently selected and are H or
C1-C3 alkyls, R3 and R4 are independently selected
and are alkyl groups having from about 10 to about 20 carbon atoms, and
at least one of R3 and R4 comprises at least two sites of
unsaturation. In certain instances, R3 and R4 are both the
same, i.e., R3 and R4 are both linoleyl (C18), etc. In
certain other instances, R3 and R4 are different, i.e., R3
is tetradectrienyl (C14) and R4 is linoleyl (C18). In a
preferred embodiment, the cationic lipid of Formula I is symmetrical,
i.e., R3 and R4 are both the same. In another preferred
embodiment, both R3 and R4 comprise at least two sites of
unsaturation. In some embodiments, R3 and R4 are independently
selected from the group consisting of dodecadienyl, tetradecadienyl,
hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment,
R3 and R4 are both linoleyl. In some embodiments, R3 and
R4 comprise at least three sites of unsaturation and are
independently selected from, e.g., dodecatrienyl, tetradectrienyl,
hexadecatrienyl, linolenyl, and icosatrienyl. In particularly preferred
embodiments, the cationic lipid of Formula I is
1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or
1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0257] Furthermore, cationic lipids of Formula II having the following
structures are useful in the present invention.

##STR00002##

wherein R1 and R2 are independently selected and are H or
C1-C3 alkyls, R3 and R4 are independently selected
and are alkyl groups having from about 10 to about 20 carbon atoms, and
at least one of R3 and R4 comprises at least two sites of
unsaturation. In certain instances, R3 and R4 are both the
same, i.e., R3 and R4 are both linoleyl (C18), etc. In
certain other instances, R3 and R4 are different, i.e., R3
is tetradectrienyl (C14) and R4 is linoleyl (C18). In a
preferred embodiment, the cationic lipids of the present invention are
symmetrical, i.e., R3 and R4 are both the same. In another
preferred embodiment, both R3 and R4 comprise at least two
sites of unsaturation. In some embodiments, R3 and R4 are
independently selected from the group consisting of dodecadienyl,
tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In a
preferred embodiment, R3 and R4 are both linoleyl. In some
embodiments, R3 and R4 comprise at least three sites of
unsaturation and are independently selected from, e.g., dodecatrienyl,
tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0258] Moreover, cationic lipids of Formula III having the following
structures (or salts thereof) are useful in the present invention.

##STR00003##

Wherein R1 and R2 are either the same or different and
independently optionally substituted C12-C24 alkyl, optionally
substituted C12-C24 alkenyl, optionally substituted
C12-C24 alkynyl, or optionally substituted C12-C24
acyl; R3 and R4 are either the same or different and
independently optionally substituted C1-C6 alkyl, optionally
substituted C1-C6 alkenyl, or optionally substituted
C1-C6 alkynyl or R3 and R4 may join to form an
optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or
2 heteroatoms chosen from nitrogen and oxygen; R5 is either absent
or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n,
and p are either the same or different and independently either 0 or 1
with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2,
3, or 4; and Y and Z are either the same or different and independently
O, S, or NH.

[0260] The cationic lipid typically comprises from about 50 mol % to about
90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to
about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol
% to about 70 mol %, from about 50 mol % to about 65 mol %, or from about
55 mol % to about 65 mol % of the total lipid present in the particle.

[0261] It will be readily apparent to one of skill in the art that
depending on the intended use of the particles, the proportions of the
components can be varied and the delivery efficiency of a particular
formulation can be measured using, e.g., an endosomal release parameter
(ERP) assay.

[0262] B. Non-Cationic Lipids

[0263] The non-cationic lipids used in the lipid particles of the
invention (e.g., SNALP) can be any of a variety of neutral uncharged,
zwitterionic, or anionic lipids capable of producing a stable complex.

[0266] In some embodiments, the non-cationic lipid present in the lipid
particles (e.g., SNALP) comprises or consists of cholesterol or a
derivative thereof, e.g., a phospholipid-free lipid particle formulation.
In other embodiments, the non-cationic lipid present in the lipid
particles (e.g., SNALP) comprises or consists of one or more
phospholipids, e.g., a cholesterol-free lipid particle formulation. In
further embodiments, the non-cationic lipid present in the lipid
particles (e.g., SNALP) comprises or consists of a mixture of one or more
phospholipids and cholesterol or a derivative thereof.

[0268] In some embodiments, the non-cationic lipid comprises from about 13
mol % to about 49.5 mol %, from about 20 mol % to about 45 mol %, from
about 25 mol % to about 45 mol %, from about 30 mol % to about 45 mol %,
from about 35 mol % to about 45 mol %, from about 20 mol % to about 40
mol %, from about 25 mol % to about 40 mol %, or from about 30 mol % to
about 40 mol % of the total lipid present in the particle.

[0269] In certain embodiments, the cholesterol present in
phospholipid-free lipid particles comprises from about 30 mol % to about
45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to
about 45 mol %, or from about 35 mol % to about 40 mol % of the total
lipid present in the particle. As a non-limiting example, a
phospholipid-free lipid particle may comprise cholesterol at about 37 mol
% of the total lipid present in the particle.

[0270] In certain other embodiments, the cholesterol present in lipid
particles containing a mixture of phospholipid and cholesterol comprises
from about 30 mol % to about 40 mol %, from about 30 mol % to about 35
mol %, or from about 35 mol % to about 40 mol % of the total lipid
present in the particle. As a non-limiting example, a lipid particle
comprising a mixture of phospholipid and cholesterol may comprise
cholesterol at about 34 mol % of the total lipid present in the particle.

[0271] In further embodiments, the cholesterol present in lipid particles
containing a mixture of phospholipid and cholesterol comprises from about
10 mol % to about 30 mol %, from about 15 mol % to about 25 mol %, or
from about 17 mol % to about 23 mol % of the total lipid present in the
particle. As a non-limiting example, a lipid particle comprising a
mixture of phospholipid and cholesterol may comprise cholesterol at about
20 mol % of the total lipid present in the particle.

[0272] In embodiments where the lipid particles contain a mixture of
phospholipid and cholesterol or a cholesterol derivative, the mixture may
comprise up to about 40, 45, 50, 55, or 60 mol % of the total lipid
present in the particle. In certain instances, the phospholipid component
in the mixture may comprise from about 2 mol % to about 12 mol %, from
about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol %,
from about 5 mol % to about 9 mol %, or from about 6 mol % to about 8 mol
% of the total lipid present in the particle. As a non-limiting example,
a lipid particle comprising a mixture of phospholipid and cholesterol may
comprise a phospholipid such as DPPC or DSPC at about 7 mol % (e.g., in a
mixture with about 34 mol % cholesterol) of the total lipid present in
the particle. In certain other instances, the phospholipid component in
the mixture may comprise from about 10 mol % to about 30 mol %, from
about 15 mol % to about 25 mol %, or from about 17 mol % to about 23 mol
% of the total lipid present in the particle. As another non-limiting
example, a lipid particle comprising a mixture of phospholipid and
cholesterol may comprise a phospholipid such as DPPC or DSPC at about 20
mol % (e.g., in a mixture with about 20 mol % cholesterol) of the total
lipid present in the particle.

[0273] C. Lipid Conjugate

[0274] In addition to cationic and non-cationic lipids, the lipid
particles of the invention (e.g., SNALP) comprise a lipid conjugate. The
conjugated lipid is useful in that it prevents the aggregation of
particles. Suitable conjugated lipids include, but are not limited to,
PEG-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid
conjugates (CPLs), and mixtures thereof. In certain embodiments, the
particles comprise either a PEG-lipid conjugate or an ATTA-lipid
conjugate together with a CPL.

[0275] In a preferred embodiment, the lipid conjugate is a PEG-lipid.
Examples of PEG-lipids include, but are not limited to, PEG coupled to
dialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No. WO
05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g.,
U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to
phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated
to ceramides as described in, e.g., U.S. Pat. No. 5,885,613, PEG
conjugated to cholesterol or a derivative thereof, and mixtures thereof.
The disclosures of these patent documents are herein incorporated by
reference in their entirety for all purposes. Additional PEG-lipids
include, without limitation, PEG-C-DOMG, 2 KPEG-DMG, and a mixture
thereof.

[0276] PEG is a linear, water-soluble polymer of ethylene PEG repeating
units with two terminal hydroxyl groups. PEGs are classified by their
molecular weights; for example, PEG 2000 has an average molecular weight
of about 2,000 daltons, and PEG 5000 has an average molecular weight of
about 5,000 daltons. PEGs are commercially available from Sigma Chemical
Co. and other companies and include, for example, the following:
monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene
glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl
succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine
(MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES),
and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other
PEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150
(e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid
conjugates of the present invention. The disclosures of these patents are
herein incorporated by reference in their entirety for all purposes. In
addition, monomethoxypolyethyleneglycol-acetic acid (MePEG-CH2COOH)
is particularly useful for preparing PEG-lipid conjugates including,
e.g., PEG-DAA conjugates.

[0277] The PEG moiety of the PEG-lipid conjugates described herein may
comprise an average molecular weight ranging from about 550 daltons to
about 10,000 daltons. In certain instances, the PEG moiety has an average
molecular weight of from about 750 daltons to about 5,000 daltons (e.g.,
from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons
to about 3,000 daltons, from about 750 daltons to about 3,000 daltons,
from about 750 daltons to about 2,000 daltons, etc.). In preferred
embodiments, the PEG moiety has an average molecular weight of about
2,000 daltons or about 750 daltons.

[0278] In certain instances, the PEG can be optionally substituted by an
alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to
the lipid or may be linked to the lipid via a linker moiety. Any linker
moiety suitable for coupling the PEG to a lipid can be used including,
e.g., non-ester containing linker moieties and ester-containing linker
moieties. In a preferred embodiment, the linker moiety is a non-ester
containing linker moiety. As used herein, the term "non-ester containing
linker moiety" refers to a linker moiety that does not contain a
carboxylic ester bond (--OC(O)--). Suitable non-ester containing linker
moieties include, but are not limited to, amido (--C(O)NH--), amino
(--NR--), carbonyl (--C(O)--), carbamate (--NHC(O)O--), urea
(--NHC(O)NH--), disulphide (--S--S--), ether (--O--), succinyl
(--(O)CCH2CH2C(O)--), succinamidyl
(--NHC(O)CH2CH2C(O)NH--), ether, disulphide, as well as
combinations thereof (such as a linker containing both a carbamate linker
moiety and an amido linker moiety). In a preferred embodiment, a
carbamate linker is used to couple the PEG to the lipid.

[0280] Phosphatidylethanolamines having a variety of acyl chain groups of
varying chain lengths and degrees of saturation can be conjugated to PEG
to form the lipid conjugate. Such phosphatidylethanolamines are
commercially available, or can be isolated or synthesized using
conventional techniques known to those of skilled in the art.
Phosphatidyl-ethanolamines containing saturated or unsaturated fatty
acids with carbon chain lengths in the range of C10 to C20 are
preferred. Phosphatidylethanolamines with mono- or diunsaturated fatty
acids and mixtures of saturated and unsaturated fatty acids can also be
used. Suitable phosphatidylethanolamines include, but are not limited to,
dimyristoyl-phosphatidylethanolamine (DMPE),
dipalmitoyl-phosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine (DOPE), and
distearoyl-phosphatidylethanolamine (DSPE).

[0281] The term "ATTA" or "polyamide" refers to, without limitation,
compounds described in U.S. Pat. Nos. 6,320,017 and 6,586,559, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes. These compounds include a compound having the
formula:

##STR00004##

wherein R is a member selected from the group consisting of hydrogen,
alkyl and acyl; R' is a member selected from the group consisting of
hydrogen and alkyl; or optionally, R and R1 and the nitrogen to
which they are bound form an azido moiety; R2 is a member of the
group selected from hydrogen, optionally substituted alkyl, optionally
substituted aryl and a side chain of an amino acid; R3 is a member
selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy,
mercapto, hydrazino, amino and NR4R5, wherein R4 and
R5 are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p
is 1 to 4; and q is 0 or 1. It will be apparent to those of skill in the
art that other polyamides can be used in the compounds of the present
invention.

[0282] The term "diacylglycerol" refers to a compound having 2 fatty acyl
chains, R' and

[0283] R2, both of which have independently between 2 and 30 carbons
bonded to the 1- and 2-position of glycerol by ester linkages. The acyl
groups can be saturated or have varying degrees of unsaturation. Suitable
acyl groups include, but are not limited to, lauryl (C12), myristyl
(C14), palmityl (C16), stearyl (C18), and icosyl
(C20). In preferred embodiments, R1 and R2 are the same,
i.e., R1 and R2 are both myristyl (i.e., dimyristyl), R1
and R2 are both stearyl (i.e., distearyl), etc. Diacylglycerols have
the following general formula:

##STR00005##

[0284] The term "dialkyloxypropyl" refers to a compound having 2 alkyl
chains, R1 and R2, both of which have independently between 2
and 30 carbons. The alkyl groups can be saturated or have varying degrees
of unsaturation. Dialkyloxypropyls have the following general formula:

##STR00006##

[0285] In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate
having the following formula:

##STR00007##

wherein R1 and R2 are independently selected and are long-chain
alkyl groups having from about 10 to about 22 carbon atoms; PEG is a
polyethyleneglycol; and L is a non-ester containing linker moiety or an
ester containing linker moiety as described above. The long-chain alkyl
groups can be saturated or unsaturated. Suitable alkyl groups include,
but are not limited to, lauryl (C12), myristyl (C14), palmityl
(C16), stearyl (C18), and icosyl (C20). In preferred
embodiments, R1 and R2 are the same, i.e., R1 and R2
are both myristyl (i.e., dimyristyl), R1 and R2 are both
stearyl (i.e., distearyl), etc.

[0286] In Formula VII above, the PEG has an average molecular weight
ranging from about 550 daltons to about 10,000 daltons. In certain
instances, the PEG has an average molecular weight of from about 750
daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about
5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from
about 750 daltons to about 3,000 daltons, from about 750 daltons to about
2,000 daltons, etc.). In preferred embodiments, the PEG has an average
molecular weight of about 2,000 daltons or about 750 daltons. The PEG can
be optionally substituted with alkyl, alkoxy, acyl, or aryl. In certain
embodiments, the terminal hydroxyl group is substituted with a methoxy or
methyl group.

[0287] In a preferred embodiment, "L" is a non-ester containing linker
moiety. Suitable non-ester containing linkers include, but are not
limited to, an amido linker moiety, an amino linker moiety, a carbonyl
linker moiety, a carbamate linker moiety, a urea linker moiety, an ether
linker moiety, a disulphide linker moiety, a succinamidyl linker moiety,
and combinations thereof. In a preferred embodiment, the non-ester
containing linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAA
conjugate). In another preferred embodiment, the non-ester containing
linker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate). In
yet another preferred embodiment, the non-ester containing linker moiety
is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

[0288] In particular embodiments, the PEG-lipid conjugate is selected
from:

##STR00008##

[0289] The PEG-DAA conjugates are synthesized using standard techniques
and reagents known to those of skill in the art. It will be recognized
that the PEG-DAA conjugates will contain various amide, amine, ether,
thio, carbamate, and urea linkages. Those of skill in the art will
recognize that methods and reagents for forming these bonds are well
known and readily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY
(Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989);
and Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.
(Longman 1989). It will also be appreciated that any functional groups
present may require protection and deprotection at different points in
the synthesis of the PEG-DAA conjugates. Those of skill in the art will
recognize that such techniques are well known. See, e.g., Green and Wuts,
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

[0290] Preferably, the PEG-DAA conjugate is a dilauryloxypropyl
(C12)-PEG conjugate, dimyristyloxypropyl (C14)-PEG conjugate, a
dipalmityloxypropyl (C16)-PEG conjugate, or a distearyloxypropyl
(C18)-PEG conjugate. Those of skill in the art will readily
appreciate that other dialkyloxypropyls can be used in the PEG-DAA
conjugates of the present invention.

[0291] In addition to the foregoing, it will be readily apparent to those
of skill in the art that other hydrophilic polymers can be used in place
of PEG. Examples of suitable polymers that can be used in place of PEG
include, but are not limited to, polyvinylpyrrolidone,
polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl
methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic
acid, polyglycolic acid, and derivatized celluloses such as
hydroxymethylcellulose or hydroxyethylcellulose.

[0292] In addition to the foregoing components, the particles (e.g., SNALP
or SPLP) of the present invention can further comprise cationic
poly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al.,
Bioconj. Chem., 11:433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use
in the present invention, and methods of making and using SPLPs and
SPLP-CPLs, are disclosed, e.g., in U.S. Pat. No. 6,852,334 and PCT
Publication No. WO 00/62813, the disclosures of which are herein
incorporated by reference in their entirety for all purposes.

[0293] Suitable CPLs include compounds of Formula VIII:

A-W-Y (VIII),

wherein A, W, and Y are as described below.

[0294] With reference to Formula VIII, "A" is a lipid moiety such as an
amphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts as a
lipid anchor. Suitable lipid examples include, but are not limited to,
diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos,
1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

[0295] "W" is a polymer or an oligomer such as a hydrophilic polymer or
oligomer. Preferably, the hydrophilic polymer is a biocompatable polymer
that is nonimmunogenic or possesses low inherent immunogenicity.
Alternatively, the hydrophilic polymer can be weakly antigenic if used
with appropriate adjuvants. Suitable nonimmunogenic polymers include, but
are not limited to, PEG, polyamides, polylactic acid, polyglycolic acid,
polylactic acid/polyglycolic acid copolymers, and combinations thereof.
In a preferred embodiment, the polymer has a molecular weight of from
about 250 to about 7,000 daltons.

[0296] "Y" is a polycationic moiety. The term polycationic moiety refers
to a compound, derivative, or functional group having a positive charge,
preferably at least 2 positive charges at a selected pH, preferably
physiological pH. Suitable polycationic moieties include basic amino
acids and their derivatives such as arginine, asparagine, glutamine,
lysine, and histidine; spermine; spermidine; cationic dendrimers;
polyamines; polyamine sugars; and amino polysaccharides. The polycationic
moieties can be linear, such as linear tetralysine, branched or
dendrimeric in structure. Polycationic moieties have between about 2 to
about 15 positive charges, preferably between about 2 to about 12
positive charges, and more preferably between about 2 to about 8 positive
charges at selected pH values. The selection of which polycationic moiety
to employ may be determined by the type of particle application which is
desired.

[0297] The charges on the polycationic moieties can be either distributed
around the entire particle moiety, or alternatively, they can be a
discrete concentration of charge density in one particular area of the
particle moiety e.g., a charge spike. If the charge density is
distributed on the particle, the charge density can be equally
distributed or unequally distributed. All variations of charge
distribution of the polycationic moiety are encompassed by the present
invention.

[0298] The lipid "A" and the nonimmunogenic polymer "W" can be attached by
various methods and preferably by covalent attachment. Methods known to
those of skill in the art can be used for the covalent attachment of "A"
and "W." Suitable linkages include, but are not limited to, amide, amine,
carboxyl, carbonate, carbamate, ester, and hydrazone linkages. It will be
apparent to those skilled in the art that "A" and "W" must have
complementary functional groups to effectuate the linkage. The reaction
of these two groups, one on the lipid and the other on the polymer, will
provide the desired linkage. For example, when the lipid is a
diacylglycerol and the terminal hydroxyl is activated, for instance with
NHS and DCC, to form an active ester, and is then reacted with a polymer
which contains an amino group, such as with a polyamide (see, e.g., U.S.
Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are herein
incorporated by reference in their entirety for all purposes), an amide
bond will form between the two groups.

[0299] In certain instances, the polycationic moiety can have a ligand
attached, such as a targeting ligand or a chelating moiety for complexing
calcium. Preferably, after the ligand is attached, the cationic moiety
maintains a positive charge. In certain instances, the ligand that is
attached has a positive charge. Suitable ligands include, but are not
limited to, a compound or device with a reactive functional group and
include lipids, amphipathic lipids, carrier compounds, bioaffinity
compounds, biomaterials, biopolymers, biomedical devices, analytically
detectable compounds, therapeutically active compounds, enzymes,
peptides, proteins, antibodies, immune stimulators, radiolabels,
fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes,
virosomes, micelles, immunoglobulins, functional groups, other targeting
moieties, or toxins.

[0300] The lipid conjugate (e.g., PEG-lipid) typically comprises from
about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %,
from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9
mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to
about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9
mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol %, from
about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about 1.8 mol
%, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about
1.6 mol %, or from about 1.4 mol % to about 1.5 mol % of the total lipid
present in the particle.

[0301] One of ordinary skill in the art will appreciate that the
concentration of the lipid conjugate can be varied depending on the lipid
conjugate employed and the rate at which the nucleic acid-lipid particle
is to become fusogenic.

[0302] By controlling the composition and concentration of the lipid
conjugate, one can control the rate at which the lipid conjugate
exchanges out of the nucleic acid-lipid particle and, in turn, the rate
at which the nucleic acid-lipid particle becomes fusogenic. For instance,
when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate
is used as the lipid conjugate, the rate at which the nucleic acid-lipid
particle becomes fusogenic can be varied, for example, by varying the
concentration of the lipid conjugate, by varying the molecular weight of
the PEG, or by varying the chain length and degree of saturation of the
acyl chain groups on the phosphatidylethanolamine or the ceramide. In
addition, other variables including, for example, pH, temperature, ionic
strength, etc. can be used to vary and/or control the rate at which the
nucleic acid-lipid particle becomes fusogenic. Other methods which can be
used to control the rate at which the nucleic acid-lipid particle becomes
fusogenic will become apparent to those of skill in the art upon reading
this disclosure.

VI. Preparation of Lipid Particles

[0303] The lipid particles of the present invention, e.g., SNALP, in which
an active agent or therapeutic agent such as an interfering RNA is
encapsulated in a lipid bilayer and is protected from degradation, can be
formed by any method known in the art including, but not limited to, a
continuous mixing method or a direct dilution process.

[0305] In certain embodiments, the present invention provides for SNALP
produced via a continuous mixing method, e.g., a process that includes
providing an aqueous solution comprising a nucleic acid such as an
interfering RNA in a first reservoir, providing an organic lipid solution
in a second reservoir, and mixing the aqueous solution with the organic
lipid solution such that the organic lipid solution mixes with the
aqueous solution so as to substantially instantaneously produce a
liposome encapsulating the nucleic acid (e.g., interfering RNA). This
process and the apparatus for carrying this process are described in
detail in U.S. Patent Publication No. 20040142025, the disclosure of
which is herein incorporated by reference in its entirety for all
purposes.

[0306] The action of continuously introducing lipid and buffer solutions
into a mixing environment, such as in a mixing chamber, causes a
continuous dilution of the lipid solution with the buffer solution,
thereby producing a liposome substantially instantaneously upon mixing.
As used herein, the phrase "continuously diluting a lipid solution with a
buffer solution" (and variations) generally means that the lipid solution
is diluted sufficiently rapidly in a hydration process with sufficient
force to effectuate vesicle generation. By mixing the aqueous solution
comprising a nucleic acid with the organic lipid solution, the organic
lipid solution undergoes a continuous stepwise dilution in the presence
of the buffer solution (i.e., aqueous solution) to produce a nucleic
acid-lipid particle.

[0307] The SNALP formed using the continuous mixing method typically have
a size of from about 40 nm to about 150 nm, from about 50 nm to about 150
nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm,
or from about 70 nm to about 90 nm. The particles thus formed do not
aggregate and are optionally sized to achieve a uniform particle size.

[0308] In another embodiment, the present invention provides for SNALP
produced via a direct dilution process that includes forming a liposome
solution and immediately and directly introducing the liposome solution
into a collection vessel containing a controlled amount of dilution
buffer. In preferred aspects, the collection vessel includes one or more
elements configured to stir the contents of the collection vessel to
facilitate dilution. In one aspect, the amount of dilution buffer present
in the collection vessel is substantially equal to the volume of liposome
solution introduced thereto. As a non-limiting example, a liposome
solution in 45% ethanol when introduced into the collection vessel
containing an equal volume of dilution buffer will advantageously yield
smaller particles.

[0309] In yet another embodiment, the present invention provides for SNALP
produced via a direct dilution process in which a third reservoir
containing dilution buffer is fluidly coupled to a second mixing region.
In this embodiment, the liposome solution formed in a first mixing region
is immediately and directly mixed with dilution buffer in the second
mixing region. In preferred aspects, the second mixing region includes a
T-connector arranged so that the liposome solution and the dilution
buffer flows meet as opposing 180° flows; however, connectors
providing shallower angles can be used, e.g., from about 27° to
about 180°. A pump mechanism delivers a controllable flow of
buffer to the second mixing region. In one aspect, the flow rate of
dilution buffer provided to the second mixing region is controlled to be
substantially equal to the flow rate of liposome solution introduced
thereto from the first mixing region. This embodiment advantageously
allows for more control of the flow of dilution buffer mixing with the
liposome solution in the second mixing region, and therefore also the
concentration of liposome solution in buffer throughout the second mixing
process. Such control of the dilution buffer flow rate advantageously
allows for small particle size formation at reduced concentrations.

[0310] These processes and the apparatuses for carrying out these direct
dilution processes are described in detail in U.S. Patent Publication No.
20070042031, the disclosure of which is herein incorporated by reference
in its entirety for all purposes.

[0311] The SNALP formed using the direct dilution process typically have a
size of from about 40 nm to about 150 nm, from about 50 nm to about 150
nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm,
or from about 70 nm to about 90 nm. The particles thus formed do not
aggregate and are optionally sized to achieve a uniform particle size.

[0312] If needed, the lipid particles of the invention (e.g., SNALP) can
be sized by any of the methods available for sizing liposomes. The sizing
may be conducted in order to achieve a desired size range and relatively
narrow distribution of particle sizes.

[0313] Several techniques are available for sizing the particles to a
desired size. One sizing method, used for liposomes and equally
applicable to the present particles, is described in U.S. Pat. No.
4,737,323, the disclosure of which is herein incorporated by reference in
its entirety for all purposes. Sonicating a particle suspension either by
bath or probe sonication produces a progressive size reduction down to
particles of less than about 50 nm in size. Homogenization is another
method which relies on shearing energy to fragment larger particles into
smaller ones. In a typical homogenization procedure, particles are
recirculated through a standard emulsion homogenizer until selected
particle sizes, typically between about 60 and about 80 nm, are observed.
In both methods, the particle size distribution can be monitored by
conventional laser-beam particle size discrimination, or QELS.

[0314] Extrusion of the particles through a small-pore polycarbonate
membrane or an asymmetric ceramic membrane is also an effective method
for reducing particle sizes to a relatively well-defined size
distribution. Typically, the suspension is cycled through the membrane
one or more times until the desired particle size distribution is
achieved. The particles may be extruded through successively smaller-pore
membranes, to achieve a gradual reduction in size.

[0315] In some embodiments, the nucleic acids in the SNALP are
precondensed as described in, e.g., U.S. patent application Ser. No.
09/744,103, the disclosure of which is herein incorporated by reference
in its entirety for all purposes.

[0316] In other embodiments, the methods will further comprise adding
non-lipid polycations which are useful to effect the lipofection of cells
using the present compositions. Examples of suitable non-lipid
polycations include, hexadimethrine bromide (sold under the brandname
POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other
salts of hexadimethrine. Other suitable polycations include, for example,
salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,
polyallylamine, and polyethyleneimine. Addition of these salts is
preferably after the particles have been formed.

[0317] In some embodiments, the nucleic acid to lipid ratios (mass/mass
ratios) in a formed SNALP will range from about 0.01 to about 0.2, from
about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01
to about 0.08. The ratio of the starting materials also falls within this
range. In other embodiments, the SNALP preparation uses about 400 μg
nucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratio
of about 0.01 to about 0.08 and, more preferably, about 0.04, which
corresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. In
other preferred embodiments, the particle has a nucleic acid:lipid mass
ratio of about 0.08.

[0318] In other embodiments, the lipid to nucleic acid ratios (mass/mass
ratios) in a formed SNALP will range from about 1 (1:1) to about 100
(100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to
about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3
(3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from
about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1),
from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25
(25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to
about 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5
(5:1) to about 15 (15:1), from about 5 (5:1) to about 10 (10:1), about 5
(5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9 (9:1), (10:1), 11 (11:1), 12 (12:1),
13 (13:1), 14 (14:1), or 15 (15:1). The ratio of the starting materials
also falls within this range.

[0319] As previously discussed, the conjugated lipid may further include a
CPL. A variety of general methods for making SNALP-CPLs (CPL-containing
SNALP) are discussed herein. Two general techniques include
"post-insertion" technique, that is, insertion of a CPL into, for
example, a pre-formed SNALP, and the "standard" technique, wherein the
CPL is included in the lipid mixture during, for example, the SNALP
formation steps. The post-insertion technique results in SNALP having
CPLs mainly in the external face of the SNALP bilayer membrane, whereas
standard techniques provide SNALP having CPLs on both internal and
external faces. The method is especially useful for vesicles made from
phospholipids (which can contain cholesterol) and also for vesicles
containing PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making
SNALP-CPL, are taught, for example, in U.S. Pat. Nos. 5,705,385;
6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication
No. 20020072121; and PCT Publication No. WO 00/62813, the disclosures of
which are herein incorporated by reference in their entirety for all
purposes.

VII. Kits

[0320] The present invention also provides lipid particles (e.g., SNALP)
in kit form. The kit may comprise a container which is compartmentalized
for holding the various elements of the lipid particles (e.g., the active
agents or therapeutic agents such as nucleic acids and the individual
lipid components of the particles). In some embodiments, the kit may
further comprise an endosomal membrane destabilizer (e.g., calcium ions).
The kit typically contains the lipid particle compositions of the present
invention, preferably in dehydrated form, with instructions for their
rehydration and administration.

[0321] As explained herein, the lipid particles of the invention (e.g.,
SNALP) can be tailored to preferentially target particular tissues,
organs, or tumors of interest. In certain instances, preferential
targeting of lipid particles such as SNALP may be carried out by
controlling the composition of the particle itself. For instance, as set
forth in Example 11, it has been found that the 1:57 PEG-cDSA SNALP
formulation can be used to preferentially target tumors outside of the
liver, whereas the 1:57 PEG-cDMA SNALP formulation can be used to
preferentially target the liver (including liver tumors).

[0322] In certain other instances, it may be desirable to have a targeting
moiety attached to the surface of the lipid particle to further enhance
the targeting of the particle. Methods of attaching targeting moieties
(e.g., antibodies, proteins, etc.) to lipids (such as those used in the
present particles) are known to those of skill in the art.

VII. Administration of Lipid Particles

[0323] Once formed, the lipid particles of the invention (e.g., SNALP) are
useful for the introduction of active agents or therapeutic agents (e.g.,
nucleic acids such as interfering RNA) into cells. Accordingly, the
present invention also provides methods for introducing an active agent
or therapeutic agent such as a nucleic acid (e.g., interfering RNA) into
a cell. The methods are carried out in vitro or in vivo by first forming
the particles as described above and then contacting the particles with
the cells for a period of time sufficient for delivery of the active
agent or therapeutic agent to the cells to occur.

[0324] The lipid particles of the invention (e.g., SNALP) can be adsorbed
to almost any cell type with which they are mixed or contacted. Once
adsorbed, the particles can either be endocytosed by a portion of the
cells, exchange lipids with cell membranes, or fuse with the cells.
Transfer or incorporation of the active agent or therapeutic agent (e.g.,
nucleic acid) portion of the particle can take place via any one of these
pathways. In particular, when fusion takes place, the particle membrane
is integrated into the cell membrane and the contents of the particle
combine with the intracellular fluid.

[0325] The lipid particles of the invention (e.g., SNALP) can be
administered either alone or in a mixture with a
pharmaceutically-acceptable carrier (e.g., physiological saline or
phosphate buffer) selected in accordance with the route of administration
and standard pharmaceutical practice. Generally, normal buffered saline
(e.g., 135-150 mM NaCl) will be employed as the
pharmaceutically-acceptable carrier. Other suitable carriers include,
e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like,
including glycoproteins for enhanced stability, such as albumin,
lipoprotein, globulin, etc. Additional suitable carriers are described
in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company,
Philadelphia, Pa., 17th ed. (1985). As used herein, "carrier" includes
any and all solvents, dispersion media, vehicles, coatings, diluents,
antibacterial and antifungal agents, isotonic and absorption delaying
agents, buffers, carrier solutions, suspensions, colloids, and the like.
The phrase "pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward reaction
when administered to a human.

[0326] The pharmaceutically-acceptable carrier is generally added
following particle formation. Thus, after the particle is formed, the
particle can be diluted into pharmaceutically-acceptable carriers such as
normal buffered saline.

[0327] The concentration of particles in the pharmaceutical formulations
can vary widely, i.e., from less than about 0.05%, usually at or at least
about 2 to 5%, to as much as about 10 to 90% by weight, and will be
selected primarily by fluid volumes, viscosities, etc., in accordance
with the particular mode of administration selected. For example, the
concentration may be increased to lower the fluid load associated with
treatment. This may be particularly desirable in patients having
atherosclerosis-associated congestive heart failure or severe
hypertension. Alternatively, particles composed of irritating lipids may
be diluted to low concentrations to lessen inflammation at the site of
administration.

[0328] The pharmaceutical compositions of the present invention may be
sterilized by conventional, well-known sterilization techniques. Aqueous
solutions can be packaged for use or filtered under aseptic conditions
and lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration. The compositions can
contain pharmaceutically-acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents and the like, for example, sodium
acetate, sodium lactate, sodium chloride, potassium chloride, and calcium
chloride. Additionally, the particle suspension may include
lipid-protective agents which protect lipids against free-radical and
lipid-peroxidative damages on storage. Lipophilic free-radical quenchers,
such as alphatocopherol and water-soluble iron-specific chelators, such
as ferrioxamine, are suitable.

[0329] A. In Vivo Administration

[0330] Systemic delivery for in vivo therapy, e.g., delivery of a
therapeutic nucleic acid to a distal target cell via body systems such as
the circulation, has been achieved using nucleic acid-lipid particles
such as those described in PCT Publication Nos. WO 05/007196, WO
05/121348, WO 05/120152, and WO 04/002453, the disclosures of which are
herein incorporated by reference in their entirety for all purposes. The
present invention also provides fully encapsulated lipid particles that
protect the nucleic acid from nuclease degradation in serum, are
nonimmunogenic, are small in size, and are suitable for repeat dosing.

[0331] For in vivo administration, administration can be in any manner
known in the art, e.g., by injection, oral administration, inhalation
(e.g., intransal or intratracheal), transdermal application, or rectal
administration. Administration can be accomplished via single or divided
doses. The pharmaceutical compositions can be administered parenterally,
i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously,
or intramuscularly. In some embodiments, the pharmaceutical compositions
are administered intravenously or intraperitoneally by a bolus injection
(see, e.g., U.S. Pat. No. 5,286,634). Intracellular nucleic acid delivery
has also been discussed in Straubringer et al., Methods Enzymol., 101:512
(1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al.,
Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem.
Res., 26:274 (1993). Still other methods of administering lipid-based
therapeutics are described in, for example, U.S. Pat. Nos. 3,993,754;
4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid
particles can be administered by direct injection at the site of disease
or by injection at a site distal from the site of disease (see, e.g.,
Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York.
pp. 70-71 (1994)). The disclosures of the above-described references are
herein incorporated by reference in their entirety for all purposes.

[0332] The compositions of the present invention, either alone or in
combination with other suitable components, can be made into aerosol
formulations (i.e., they can be "nebulized") to be administered via
inhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,
Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed into
pressurized acceptable propellants, such as dichlorodifluoromethane,
propane, nitrogen, and the like.

[0333] In certain embodiments, the pharmaceutical compositions may be
delivered by intranasal sprays, inhalation, and/or other aerosol delivery
vehicles. Methods for delivering nucleic acid compositions directly to
the lungs via nasal aerosol sprays have been described, e.g., in U.S.
Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs using
intranasal microparticle resins and lysophosphatidyl-glycerol compounds
(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceutical arts.
Similarly, transmucosal drug delivery in the form of a
polytetrafluoroetheylene support matrix is described in U.S. Pat. No.
5,780,045. The disclosures of the above-described patents are herein
incorporated by reference in their entirety for all purposes.

[0334] Formulations suitable for parenteral administration, such as, for
example, by intraarticular (in the joints), intravenous, intramuscular,
intradermal, intraperitoneal, and subcutaneous routes, include aqueous
and non-aqueous, isotonic sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions that can include suspending
agents, solubilizers, thickening agents, stabilizers, and preservatives.
In the practice of this invention, compositions are preferably
administered, for example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically, or intrathecally.

[0335] Generally, when administered intravenously, the lipid particle
formulations are formulated with a suitable pharmaceutical carrier. Many
pharmaceutically acceptable carriers may be employed in the compositions
and methods of the present invention. Suitable formulations for use in
the present invention are found, for example, in REMINGTON'S
PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17th
ed. (1985). A variety of aqueous carriers may be used, for example,
water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may
include glycoproteins for enhanced stability, such as albumin,
lipoprotein, globulin, etc. Generally, normal buffered saline (135-150 mM
NaCl) will be employed as the pharmaceutically acceptable carrier, but
other suitable carriers will suffice. These compositions can be
sterilized by conventional liposomal sterilization techniques, such as
filtration. The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting agents,
wetting agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc. These compositions can be
sterilized using the techniques referred to above or, alternatively, they
can be produced under sterile conditions. The resulting aqueous solutions
may be packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a sterile
aqueous solution prior to administration.

[0336] In certain applications, the lipid particles disclosed herein may
be delivered via oral administration to the individual. The particles may
be incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,
mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,
e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosures
of which are herein incorporated by reference in their entirety for all
purposes). These oral dosage forms may also contain the following:
binders, gelatin; excipients, lubricants, and/or flavoring agents. When
the unit dosage form is a capsule, it may contain, in addition to the
materials described above, a liquid carrier. Various other materials may
be present as coatings or to otherwise modify the physical form of the
dosage unit. Of course, any material used in preparing any unit dosage
form should be pharmaceutically pure and substantially non-toxic in the
amounts employed.

[0337] Typically, these oral formulations may contain at least about 0.1%
of the lipid particles or more, although the percentage of the particles
may, of course, be varied and may conveniently be between about 1% or 2%
and about 60% or 70% or more of the weight or volume of the total
formulation. Naturally, the amount of particles in each therapeutically
useful composition may be prepared is such a way that a suitable dosage
will be obtained in any given unit dose of the compound. Factors such as
solubility, bioavailability, biological half-life, route of
administration, product shelf life, as well as other pharmacological
considerations will be contemplated by one skilled in the art of
preparing such pharmaceutical formulations, and as such, a variety of
dosages and treatment regimens may be desirable.

[0338] Formulations suitable for oral administration can consist of: (a)
liquid solutions, such as an effective amount of a packaged therapeutic
agent such as nucleic acid (e.g., interfering RNA) suspended in diluents
such as water, saline, or PEG 400; (b) capsules, sachets, or tablets,
each containing a predetermined amount of a therapeutic agent such as
nucleic acid (e.g., interfering RNA), as liquids, solids, granules, or
gelatin; (c) suspensions in an appropriate liquid; and (d) suitable
emulsions. Tablet forms can include one or more of lactose, sucrose,
mannitol, sorbitol, calcium phosphates, corn starch, potato starch,
microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,
magnesium stearate, stearic acid, and other excipients, colorants,
fillers, binders, diluents, buffering agents, moistening agents,
preservatives, flavoring agents, dyes, disintegrating agents, and
pharmaceutically compatible carriers. Lozenge forms can comprise a
therapeutic agent such as nucleic acid (e.g., interfering RNA) in a
flavor, e.g., sucrose, as well as pastilles comprising the therapeutic
agent in an inert base, such as gelatin and glycerin or sucrose and
acacia emulsions, gels, and the like containing, in addition to the
therapeutic agent, carriers known in the art.

[0339] In another example of their use, lipid particles can be
incorporated into a broad range of topical dosage forms. For instance, a
suspension containing nucleic acid-lipid particles such as SNALP can be
formulated and administered as gels, oils, emulsions, topical creams,
pastes, ointments, lotions, foams, mousses, and the like.

[0340] When preparing pharmaceutical preparations of the lipid particles
of the invention, it is preferable to use quantities of the particles
which have been purified to reduce or eliminate empty particles or
particles with therapeutic agents such as nucleic acid associated with
the external surface.

[0341] The methods of the present invention may be practiced in a variety
of hosts. Preferred hosts include mammalian species, such as primates
(e.g., humans and chimpanzees as well as other nonhuman primates),
canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats
and mice), lagomorphs, and swine.

[0342] The amount of particles administered will depend upon the ratio of
therapeutic agent (e.g., nucleic acid) to lipid, the particular
therapeutic agent (e.g., nucleic acid) used, the disease or disorder
being treated, the age, weight, and condition of the patient, and the
judgment of the clinician, but will generally be between about 0.01 and
about 50 mg per kilogram of body weight, preferably between about 0.1 and
about 5 mg/kg of body weight, or about 108-1010 particles per
administration (e.g., injection).

[0343] B. In Vitro Administration

[0344] For in vitro applications, the delivery of therapeutic agents such
as nucleic acids (e.g., interfering RNA) can be to any cell grown in
culture, whether of plant or animal origin, vertebrate or invertebrate,
and of any tissue or type. In preferred embodiments, the cells are animal
cells, more preferably mammalian cells, and most preferably human cells.

[0345] Contact between the cells and the lipid particles, when carried out
in vitro, takes place in a biologically compatible medium. The
concentration of particles varies widely depending on the particular
application, but is generally between about 1 μmol and about 10 mmol.
Treatment of the cells with the lipid particles is generally carried out
at physiological temperatures (about 37° C.) for periods of time
of from about 1 to 48 hours, preferably of from about 2 to 4 hours.

[0346] In one group of preferred embodiments, a lipid particle suspension
is added to 60-80% confluent plated cells having a cell density of from
about 103 to about 105 cells/ml, more preferably about
2×104 cells/ml. The concentration of the suspension added to
the cells is preferably of from about 0.01 to 0.2 μg/ml, more
preferably about 0.1 μg/ml.

[0347] Using an Endosomal Release Parameter (ERP) assay, the delivery
efficiency of the SNALP or other lipid particle of the invention can be
optimized. An ERP assay is described in detail in U.S. Patent Publication
No. 20030077829, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. More particularly, the
purpose of an ERP assay is to distinguish the effect of various cationic
lipids and helper lipid components of SNALP based on their relative
effect on binding/uptake or fusion with/destabilization of the endosomal
membrane. This assay allows one to determine quantitatively how each
component of the SNALP or other lipid particle affects delivery
efficiency, thereby optimizing the SNALP or other lipid particle.
Usually, an ERP assay measures expression of a reporter protein (e.g.,
luciferase, β-galactosidase, green fluorescent protein (GFP), etc.),
and in some instances, a SNALP formulation optimized for an expression
plasmid will also be appropriate for encapsulating an interfering RNA. In
other instances, an ERP assay can be adapted to measure downregulation of
transcription or translation of a target sequence in the presence or
absence of an interfering RNA (e.g., siRNA). By comparing the ERPs for
each of the various SNALP or other lipid particles, one can readily
determine the optimized system, e.g., the SNALP or other lipid particle
that has the greatest uptake in the cell.

[0350] In vivo delivery of lipid particles such as SNALP encapsulating an
interfering RNA (e.g., siRNA) is suited for targeting cells of any cell
type. The methods and compositions can be employed with cells of a wide
variety of vertebrates, including mammals, such as, e.g, canines,
felines, equines, bovines, ovines, caprines, rodents (e.g., mice, rats,
and guinea pigs), lagomorphs, swine, and primates (e.g. monkeys,
chimpanzees, and humans).

[0351] To the extent that tissue culture of cells may be required, it is
well-known in the art. For example, Freshney, Culture of Animal Cells, a
Manual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchler
et al., Biochemical Methods in Cell Culture and Virology, Dowden,
Hutchinson and Ross, Inc. (1977), and the references cited therein
provide a general guide to the culture of cells. Cultured cell systems
often will be in the form of monolayers of cells, although cell
suspensions are also used.

[0352] D. Detection of Lipid Particles

[0353] In some embodiments, the lipid particles of the present invention
(e.g., SNALP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7,
8 or more hours. In other embodiments, the lipid particles of the present
invention (e.g., SNALP) are detectable in the subject at about 8, 12, 24,
48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24,
25, or 28 days after administration of the particles. The presence of the
particles can be detected in the cells, tissues, or other biological
samples from the subject. The particles may be detected, e.g., by direct
detection of the particles, detection of a therapeutic nucleic acid such
as an interfering RNA (e.g., siRNA) sequence, detection of the target
sequence of interest (i.e., by detecting expression or reduced expression
of the sequence of interest), or a combination thereof.

[0354] 1. Detection of Particles

[0355] Lipid particles of the invention such as SNALP can be detected
using any method known in the art. For example, a label can be coupled
directly or indirectly to a component of the lipid particle using methods
well-known in the art. A wide variety of labels can be used, with the
choice of label depending on sensitivity required, ease of conjugation
with the lipid particle component, stability requirements, and available
instrumentation and disposal provisions. Suitable labels include, but are
not limited to, spectral labels such as fluorescent dyes (e.g.,
fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)
and Oregon Green®; rhodamine and derivatives such Texas red,
tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,
phycoerythrin, AMCA, CyDyes®, and the like; radiolabels such as
3H, 125I, 35S, 14C, 32P, 33P, etc.; enzymes
such as horse radish peroxidase, alkaline phosphatase, etc.; spectral
colorimetric labels such as colloidal gold or colored glass or plastic
beads such as polystyrene, polypropylene, latex, etc. The label can be
detected using any means known in the art.

[0356] 2. Detection of Nucleic Acids

[0357] Nucleic acids (e.g., interfering RNA) are detected and quantified
herein by any of a number of means well-known to those of skill in the
art. The detection of nucleic acids may proceed by well-known methods
such as Southern analysis, Northern analysis, gel electrophoresis, PCR,
radiolabeling, scintillation counting, and affinity chromatography.
Additional analytic biochemical methods such as spectrophotometry,
radiography, electrophoresis, capillary electrophoresis, high performance
liquid chromatography (HPLC), thin layer chromatography (TLC), and
hyperdiffusion chromatography may also be employed.

[0358] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known to
those skilled in the art. For example, common formats include sandwich
assays and competition or displacement assays. Hybridization techniques
are generally described in, e.g., "Nucleic Acid Hybridization, A
Practical Approach," Eds. Hames and Higgins, IRL Press (1985).

[0359] The sensitivity of the hybridization assays may be enhanced through
use of a nucleic acid amplification system which multiplies the target
nucleic acid being detected. In vitro amplification techniques suitable
for amplifying sequences for use as molecular probes or for generating
nucleic acid fragments for subsequent subcloning are known. Examples of
techniques sufficient to direct persons of skill through such in vitro
amplification methods, including the polymerase chain reaction (PCR) the
ligase chain reaction (LCR), Qβ-replicase amplification and other
RNA polymerase mediated techniques (e.g., NASBA®) are found in
Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS IN
MOLECULAR BIOLOGY, eds., Current Protocols, Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc. (2002); as well as U.S. Pat. No.
4,683,202; PCR Protocols, A Guide to Methods and Applications (Innis et
al. eds.) Academic Press Inc. San Diego, Calif. (1990); Arnheim &
Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research, 3:81
(1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli
et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomeli et al., J.
Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077 (1988);
Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560
(1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan and Malek,
Biotechnology, 13:563 (1995). Improved methods of cloning in vitro
amplified nucleic acids are described in U.S. Pat. No. 5,426,039. Other
methods described in the art are the nucleic acid sequence based
amplification (NASBA®, Cangene, Mississauga, Ontario) and
Qβ-replicase systems. These systems can be used to directly identify
mutants where the PCR or LCR primers are designed to be extended or
ligated only when a select sequence is present. Alternatively, the select
sequences can be generally amplified using, for example, nonspecific PCR
primers and the amplified target region later probed for a specific
sequence indicative of a mutation. The disclosures of the above-described
references are herein incorporated by reference in their entirety for all
purposes.

[0360] Nucleic acids for use as probes, e.g., in in vitro amplification
methods, for use as gene probes, or as inhibitor components are typically
synthesized chemically according to the solid phase phosphoramidite
triester method described by Beaucage et al., Tetrahedron Letts., 22:1859
1862 (1981), e.g., using an automated synthesizer, as described in
Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984).
Purification of ploynucleotides, where necessary, is typically performed
by either native acrylamide gel electrophoresis or by anion exchange HPLC
as described in Pearson et al., J. Chrom., 255:137 149 (1983). The
sequence of the synthetic poluyucleotides can be verified using the
chemical degradation method of Maxam and Gilbert (1980) in Grossman and
Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

[0361] An alternative means for determining the level of transcription is
in situ hybridization. In situ hybridization assays are well-known and
are generally described in Angerer et al., Methods Enzymol., 152:649
(1987). In an in situ hybridization assay, cells are fixed to a solid
support, typically a glass slide. If DNA is to be probed, the cells are
denatured with heat or alkali. The cells are then contacted with a
hybridization solution at a moderate temperature to permit annealing of
specific probes that are labeled. The probes are preferably labeled with
radioisotopes or fluorescent reporters.

VIII. Examples

[0362] The present invention will be described in greater detail by way of
specific examples. The following examples are offered for illustrative
purposes, and are not intended to limit the invention in any manner.
Those of skill in the art will readily recognize a variety of noncritical
parameters which can be changed or modified to yield essentially the same
results.

Example 1

Materials and Methods

[0363] siRNA: All siRNA molecules used in these studies were chemically
synthesized by the University of Calgary (Calgary, AB) or Dharmacon Inc.
(Lafayette, Colo.). The siRNAs were desalted and annealed using standard
procedures.

[0364] Lipid Encapsulation of siRNA: In some embodiments, siRNA molecules
were encapsulated into nucleic acid-lipid particles composed of the
following lipids: the lipid conjugate PEG-cDMA
(3-N-[(-Methoxypoly(ethylene
glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); the cationic lipid
DLinDMA (1,2-Dilinoleyloxy-3-(N,N-dimethyl)aminopropane); the
phospholipid DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; Avanti
Polar Lipids; Alabaster, Ala.); and synthetic cholesterol (Sigma-Aldrich
Corp.; St. Louis, Mo.) in the molar ratio 1.4:57.1:7.1:34.3,
respectively. In other words, siRNAs were encapsulated into SNALP of the
following "1:57" formulation: 1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC;
and 34.3% cholesterol. In other embodiments, siRNA molecules were
encapsulated into phospholipid-free SNALP composed of the following
lipids: the lipid conjugate PEG-cDMA; the cationic lipid DLinDMA; and
synthetic cholesterol in the molar ratio 1.5:61.5:36.9, respectively. In
other words, siRNAs were encapsulated into phospholipid-free SNALP of the
following "1:62" formulation: 1.5% PEG-cDMA; 61.5% DLinDMA; and 36.9%
cholesterol. For vehicle controls, empty particles with identical lipid
composition were formed in the absence of siRNA. It should be understood
that the 1:57 formulation and 1:62 formulation are target formulations,
and that the amount of lipid (both cationic and non-cationic) present and
the amount of lipid conjugate present in the formulation may vary.
Typically, in the 1:57 formulation, the amount of cationic lipid will be
57 mol %±5 mol %, and the amount of lipid conjugate will be 1.5 mol
%±0.5 mol %, with the balance of the 1:57 formulation being made up of
non-cationic lipid (e.g., phospholipid, cholesterol, or a mixture of the
two). Similarly, in the 1:62 formulation, the amount of cationic lipid
will be 62 mol % t 5 mol %, and the amount of lipid conjugate will be 1.5
mol % f 0.5 mol %, with the balance of the 1:62 formulation being made up
of the non-cationic lipid (e.g., cholesterol).

[0365] SNALP formulations were prepared with an siRNA targeting Eg5 as the
nucleic acid component. Eg5 is a member of kinesin-related proteins that
are involved in functions related to movements of organelles,
microtubules, or chromosomes along microtubules. These functions include
axonal transport, microtubule sliding during nuclear fusion or division,
and chromosome disjunction during meiosis and early mitosis. Eg5 plays a
critical role in mitosis of mammalian cells. The Eg5 siRNA used in this
study is provided in Table 1. The modifications involved introducing
2'OMe-uridine at selected positions in the sense and antisense strands of
the Eg5 2263 siRNA sequence, in which the siRNA duplex contained less
than about 20% 2'OMe-modified nucleotides.

[0366] The lipid components and physical characteristics of the SNALP
formulations are summarized in Table 2. The lipid:drug ratio is described
in units of mg total lipid per mg nucleic acid. Mean particle size and
polydispersity were measured on a Malvern Instruments Zetasizer.
Encapsulation of nucleic acid was measured using a Ribogreen assay
essentially as described in Heyes et al., Journal of Controlled Release,
107:276-287 (2005).

[0367] Silencing of Eg5 by siRNA transfection causes mitotic arrest and
apoptosis in mammalian cells. Cell viability following transfection with
SNALP containing an siRNA targeting Eg5 therefore provides a simple
biological readout of in vitro transfection efficiency. Cell viability of
in vitro cell cultures was assessed using the commercial reagent
CellTiter-Blue® (Promega Corp.; Madison, Wis.), a resazurin dye that
is reduced by metabolically active cells to the fluorogenic product
resorufin. The human colon cancer cell line HT29 was cultured using
standard tissue culture techniques. 72 hours after SNALP application,
CellTiter-Blue® reagent was added to the culture to quantify the
metabolic activity of the cells, which is a measure of cell viability.
Data are presented as a percent of cell viability relative to
("untreated") control cells that received phosphate buffered saline (PBS)
vehicle only.

[0369] SNALP formulations were prepared with an siRNA targeting
apolipoprotein B (ApoB) as the nucleic acid component. ApoB is the main
apolipoprotein of chylomicrons and low density lipoproteins (LDL).
Mutations in ApoB are associated with hypercholesterolemia. ApoB occurs
in the plasma in 2 main forms, ApoB48 and ApoB 100, which are synthesized
in the intestine and liver, respectively, due to an organ-specific stop
codon. The ApoB siRNA used in this study is provided in Table 3. The
modifications involved introducing 2'OMe-uridine or 2'OMe-guanosine at
selected positions in the sense and antisense strands of the ApoB siRNA
sequence, in which the siRNA duplex contained less than about 20%
2'OMe-modified nucleotides.

[0370] The lipid components and physical characteristics of the
formulations are summarized in Table 4. The lipid:drug ratio is described
in units of mg total lipid per mg nucleic acid. Mean particle size and
polydispersity were measured on a Malvern Instruments Zetasizer.
Encapsulation of nucleic acid was measured using a Ribogreen assay
essentially as described in Heyes et al., Journal of Controlled Release,
107:276-287 (2005).

[0371] BALB/c mice (female, at least 4 weeks old) were obtained from
Harlan Labs. After an acclimation period (of at least 7 days), animals
were administered SNALP by intravenous (IV) injection in the lateral tail
vein once daily on Study Day 0 (1 dose total per animal). Dosage was 1 mg
encapsulated siRNA per kg body weight, corresponding to 10 ml/kg (rounded
to the nearest 10 μl). As a negative control, one group of animals was
given an IV injection of phosphate buffered saline (PBS) vehicle. On
Study Day 2, animals were euthanized and liver tissue was collected in
RNAlater.

[0374] SNALP formulations were prepared with the ApoB siRNA set forth in
Table 3. The lipid components and physical characteristics of the
formulations are summarized in Table 5. The lipid:drug ratio is described
in units of mg total lipid per mg nucleic acid. Mean particle size and
polydispersity were measured on a Malvern Instruments Zetasizer.
Encapsulation of nucleic acid was measured using a Ribogreen assay
essentially as described in Heyes et al., Journal of Controlled Release,
107:276-287 (2005).

[0375] The 2:30 SNALP formulation used in this study is lipid composition
2:30:20:48 as described in molar percentages of PEG-C-DMA, DLinDMA, DSPC,
and cholesterol (in that order). This formulation was prepared by syringe
press at an input lipid to drug (L:D) ratio (mg:mg) of 13:1.

[0376] The 1:57 SNALP formulation used in this study is lipid composition
1.5:57.1:7:34.3 as described in molar percentages of PEG-C-DMA, DLinDMA,
DPPC, and cholesterol (in that order). This formulation was prepared by
syringe press at an input lipid to drug (L:D) ratio (mg:mg) of 9:1.

[0377] BALB/c mice (female, 4 weeks old) were obtained from Harlan Labs.
After an acclimation period (of at least 7 days), animals were
administered SNALP by intravenous (IV) injection in the lateral tail vein
once daily on Study Days 0, 1, 2, 3 & 4 for a total of 5 doses per
animal. Daily dosage was either 1.0 (for 2:30 SNALP) or 0.1 (for 1:57
SNALP) mg encapsulated siRNA per kg body weight, corresponding to 10
ml/kg (rounded to the nearest 10 μl). As a negative control, one group
of animals was given IV injections of phosphate buffered saline (PBS)
vehicle. On Study Day 7, 72 h after the last treatment, animals were
euthanized and liver tissue was collected in RNAlater.

[0379] FIG. 3 shows that the 1:57 SNALP containing ApoB 10048 U2/2 G1/2
siRNA was more than 10 times as efficacious as the 2:30 SNALP in
mediating ApoB gene silencing in mouse liver at a 10-fold lower dose.

[0380] SNALP formulations were prepared with the ApoB siRNA set forth in
Table 3. The lipid components and physical characteristics of the
formulations are summarized in Table 6.

[0381] The lipid:drug ratio is described in units of mg total lipid per mg
nucleic acid. Mean particle size and polydispersity were measured on a
Malvern Instruments Zetasizer. Encapsulation of nucleic acid was measured
using a Ribogreen assay essentially as described in Heyes et al., Journal
of Controlled Release, 107:276-287 (2005).

[0382] BALB/c mice (female, at least 4 weeks old) were obtained from
Harlan Labs. After an acclimation period (of at least 7 days), animals
were administered SNALP by intravenous (IV) injection in the lateral tail
vein once daily on Study Day 0 (1 dose total per animal). Dosage was 0.75
mg encapsulated siRNA per kg body weight, corresponding to 10 ml/kg
(rounded to the nearest 10 μl). As a negative control, one group of
animals was given an IV injection of phosphate buffered saline (PBS)
vehicle. On Study Day 2, animals were euthanized and liver tissue was
collected in RNAlater.

[0385] SNALP formulations were prepared with the ApoB siRNA set forth in
Table 3. The lipid components and physical characteristics of the
formulations are summarized in Table 7.

[0386] The lipid:drug ratio is described in units of mg total lipid per mg
nucleic acid. Mean particle size and polydispersity were measured on a
Malvern Instruments Zetasizer. Encapsulation of nucleic acid was measured
using a Ribogreen assay essentially as described in Heyes et al., Journal
of Controlled Release, 107:276-287 (2005).

[0387] BALB/c mice (female, at least 4 weeks old) were obtained from
Harlan Labs. After an acclimation period (of at least 7 days), animals
were administered SNALP by intravenous (IV) injection in the lateral tail
vein once daily on Study Day 0 (1 dose total per animal). Dosage was 0.1
mg encapsulated siRNA per kg body weight, corresponding to 10 ml/kg
(rounded to the nearest 10 μl). As a negative control, one group of
animals was given an IV injection of phosphate buffered saline (PBS)
vehicle. On Study Day 2, animals were euthanized and liver tissue was
collected in RNAlater.

[0389] FIG. 5 shows that the 1:62 SNALP formulation was one of the most
potent inhibitors of ApoB expression at two different lipid:drug ratios
(i.e., 6.1 & 10.1) among the phospholipid-free SNALP formulations tested
(see, Groups 2 & 15).

[0390] This study illustrates a comparison of the tolerability and
efficacy of the 1:57 SNALP formulation with ApoB-targeting siRNA as
prepared by various manufacturing processes. In particular, 1:57 SNALP
was prepared by a syringe press or gear pump process using either PBS or
citrate buffer (post-blend dilution) and administered intravenously in
mice.

[0396] Formulation Details: [0397] 1. Lipid composition "1|57 Citrate
blend" used in this study is 1.4:57.1:7.1:34.3 as described in molar
percentages of PEG-C-DMA, DLinDMA, DPPC, and cholesterol (in that order).
This formulation has an input lipid to drug ratio of 8.9. [0398] 2. Gear
pump set up included 0.8 mm T-connector and 400 mL/min speed. [0399] 3.
siRNA used in this study is apoB-10048 U2/2 G1/2 siRNA.

[0401] Treatment: Just prior to the first treatment, animals are weighed
and dose amounts are calculated based on the weight of individual animals
(equivalent to 10 mL/kg, rounded to the nearest 10 μl). Test article
is administered by IV injection through the tail vein once on Day 0 (1
dose total per animal). Body weight is measured daily (every 24 h) for
the duration of the study. Cage-side observations are taken daily in
concert with body weight measurements and additionally as warranted.

[0403] Group 8-12 Endpoint: Animals are sacrificed on Day 2, 48 h after
test article administration. Blood is collected by cardiac puncture and
processed for plasma. Immediately centrifuge for 5 min at 16,000×g
(at 16° C.). Record any observations of unusual plasma appearance.
Pipette off clear plasma supernatant into a clean microfuge tube and
store at -80° C. The following tissues are removed and weighed
separately: liver and spleen. The bottom (unattached) half of the left
liver lobe is detached and submerged in >5 volumes of RNAlater
(<0.3 g in 1.5 mL RNAlater in 2.0 mL tube), stored at least 16 hours
at 4° C. prior to analysis and long term storage at -20° C.
or -80° C. for archival purposes. Formulations are expected to be
well tolerated. Mice which exhibit signs of distress associated with the
treatment are terminated at the discretion of the vivarium staff.

[0404] Termination: Mice are anaesthetized with a lethal dose of
ketamine/xylazine; then cardiac puncture is performed followed by
cervical dislocation.

[0406] There was no body weight loss or change in animal
appearance/behavior upon administration of the 1:57 SNALP formulations.
FIG. 6 shows that the tolerability of SNALP prepared by citrate buffer
versus PBS direct dilution did not differ significantly in terms of blood
clinical chemistry parameters. There was a tolerability difference
between syringe citrate and syringe PBS at constant siRNA dosage, but
that was likely an artifact dependent on the different final lipid:drug
(L:D) ratios of these two preparations.

[0407] FIG. 7 shows that the efficacy of the 1:57 SNALP prepared by gear
pump was similar to the same SNALP prepared by syringe press. The
tolerability profile was improved with the gear pump process, which could
be attributed to increased initial encapsulation rate and decreased final
L:D ratio.

[0408] This study illustrates a comparison of the tolerability and
efficacy of the 1:57 SNALP formulation with ApoB-targeting siRNA as
prepared by a direct dilution or in-line dilution process at an input
lipid to drug ratio of 6:1 or 9:1.

[0415] Formulation Details: [0416] 1. "1|57 SNALP" used in this study is
lipid composition 1.4:57.1:7.1:34.3 as described in molar percentages of
PEG-C-DMA, DLinDMA, DPPC, and cholesterol (in that order). This
formulation was prepared by gear pump at an input lipid to drug ratio of
9:1 (28 mM lipids) or 6:1 (14 mM lipids). [0417] 2. siRNA used in this
study is apoB-10048 U2/2 G1/2 siRNA.

[0419] Treatment: Just prior to the first treatment, animals are weighed
and dose amounts are calculated based on the weight of individual animals
(equivalent to 10 mL/kg, rounded to the nearest 10 μl). Test article
is administered by IV injection through the tail vein once on Day 0 (1
dose total per animal). Body weight is measured daily (every 24 h) for
the duration of the study. Cage-side observations are taken daily in
concert with body weight measurements and additionally as warranted.

[0421] Groups 1-3: Blood is collected by cardiac puncture upon sacrifice.
Whole amount is collected into an EDTA microtainer, mixed immediately to
prevent coagulation, and sent for analysis of CBC/Diff profile. Perform
brief necropsy.

[0423] Groups 12-19: Blood is collected by cardiac puncture and processed
for plasma: immediately centrifuge for 5 min at 16,000×g (at
16° C.). Record any observations of unusual plasma appearance.
Pipette off clear plasma supernatant into a clean microfuge tube and
store at -80° C. The following tissues are removed: liver. The
liver is not weighed; the bottom (unattached) half of the left liver lobe
is detached and submerged in >5 volumes of RNAlater (<0.3 g in 1.5
mL RNAlater in 2.0 mL tube), stored at least 16 hours at 4° C.
prior to analysis and long term storage at -80° C. Formulations
are expected to be well tolerated. Mice which exhibit signs of distress
associated with the treatment are terminated at the discretion of the
vivarium staff.

[0424] Termination: Mice are anaesthetized with a lethal dose of
ketamine/xylazine; then cardiac puncture is performed followed by
cervical dislocation.

[0426] Plasma ApoB-100 is measured using ELISA. Plasma total cholesterol
is measured using a standard enzymatic assay.

Results

[0427] Tolerability:

[0428] FIG. 8 shows that there was very little effect on body weight 24
hours after 1:57 SNALP administration. The maximum weight loss of
3.6±0.7% was observed at the highest drug dose of 17 mg/kg. There was
also no obvious change in animal appearance/behavior at any of the
dosages tested.

[0429]FIG. 9 shows that there were no obvious changes in platelet count.
Reduction of platelets can cause the mean platelet volume to increase as
the body produces new platelets in compensation for the treatment-related
decrease. Under the conditions of this study, the mean platelet volume
did not change in SNALP-treated groups.

[0430] FIG. 10 shows that clinically significant liver enzyme elevations
(3xULN) occurred at drug dosages of 11 mg/kg for 1:57 SNALP at a
lipid:drug (L:D) ratio of 10, and at 13 mg/kg at a L:D of 7. A slight
dose response trend upwards in plasma total protein and globulin was also
observed.

Efficacy:

[0431] FIG. 11 shows that based on the liver mRNA QuantiGene analysis, the
potency of the lower L:D SNALP was as good as that of the higher L:D
SNALP at the tested drug dosages. In fact, the ApoB silencing activity
was identical at the 0.05 and 0.1 mg/kg dosages. As such, the potency of
the 1:57 SNALP at a 6:1 input L:D ratio (final ratio of 7:1) was similar
to the potency of the 1:57 SNALP at a 9:1 input L:D ratio (final ratio of
10:1) at reducing ApoB expression.

[0432] FIG. 12 shows that ApoB protein and total cholesterol levels were
reduced to a similar extent by the 1:57 SNALP at a 6:1 input L:D ratio
(final ratio of 7:1) and the 1:57 SNALP at a 9:1 input L:D ratio (final
ratio of 10:1).

Therapeutic Index:

[0433] This study demonstrates that both the 1:57 SNALP at a 6:1 input L:D
ratio (final ratio of 7:1) and the 1:57 SNALP at a 9:1 input L:D ratio
(final ratio of 10:1) caused about 60% ApoB liver mRNA silencing with a
drug dose of 0.1 mg/kg. Interpolating from the available data points in
FIG. 10, a 10:1 final L:D ratio at 10 mg/kg may cause a similar degree of
enzyme elevation as a 7:1 final L:D ratio at 13 mg/kg. Using these
activity and toxicity points, the therapeutic index for the 1:57 SNALP at
a 10:1 final L:D ratio is (10 mg/kg)/(0.1 mg/kg)=100 and the therapeutic
index for the 1:57 SNALP at a 7:1 final L:D ratio is (13 mg/kg)/(0.1
mg/kg)=130. Using this dataset, the therapeutic index for the 1:57 SNALP
at a 7:1 final L:D ratio is 30% greater than the therapeutic index for
the 1:57 SNALP at a 10:1 final L:D ratio.

[0436] All samples were filter-sterilized prior to dilution to working
concentration. All tubes were labeled with the formulation date, lipid
composition, and nucleic acid concentration. SNALP samples were provided
at 0.2 mg/ml nucleic acid. A minimum of 20 ml of each SNALP was required
to perform the study. Formulations for this study contained:

TABLE-US-00018
[0437] Day 0 Mice will receive Anafen by SC injection (100 μg in 20
μl saline)
immediately prior to surgery. Individual mice are anesthetized by
isoflourane gas inhalation and eye lube applied to prevent excessive
eye drying. While maintained under gas anesthesia from a nose cone,
a single 1.5 cm incision across the midline will be made below the
sternum. The left lateral hepatic lobe is then exteriorized using an
autoclaved cotton wool bud. 25 μl of tumor cells suspended in PBS is
injected into the lobe at a shallow angle using a leur tip Hamilton
syringe (50 μl) and 30G (3/8'') needle. Cells will be injected slowly
(~30 s) and a swab applied to the puncture wound immediately after
needle withdrawal. After any bleeding has stopped (~1 min), the
incision is closed with 5-6 sutures in the muscle wall and 3-4 skin
clips. Cell suspensions will be thoroughly mixed immediately prior to
each injection. Mice will recover from anesthesia in a clean cage
lined with paper towel and monitored closely for 2-4 hours. Animals
are then returned to normal housing.
Day 1 All mice will be lightly anesthetized by isoflourane gas and the
sutures examined. Animals will then receive Anafen by SC injection
(100 μg in 20 μl saline).
Day 10 Mice will be randomized into the appropriate treatment groups.
Day 11 Groups A, B - Day 11: All Animals will be administered SNALP at
2 mg/kg by IV injection via the lateral tail vein. Mice will be dosed
according to body weight (10 ml/kg). Dosing will be repeated for 5
consecutive days based on initial weight.
Day 14-35 Groups A, B - Days 14, 17, 21, 25, 28, 32, 35: All Animals will
be
re-administered SNALP at 2 mg/kg by IV injection via the lateral tail
vein.
Mice will be dosed according to body weight (10 ml/kg).
Body weights Groups: Mice will be weighed on the day of dosing
for 5 weeks, then twice weekly until close of the study.
Endpoint: Tumor burden and formulations are expected to be well
tolerated. Mice that exhibit signs of distress associated with the
treatment or tumor burden are terminated at the discretion of the
vivarium staff.
Termination: Mice are anesthetized with a lethal dose of ketamine/xylazine
followed by cervical dislocation.
Data Analysis: Survival and body weights are assayed.

Results

[0438]FIG. 13 shows the mean body weights of mice during therapeutic
dosing of PLK1424 SNALP in the Hep3B intrahepatic (I.H.) tumor model. The
treatment regimen was well tolerated with no apparent signs of
treatment-related toxicity.

[0439] FIG. 14 shows that treatment with 1:57 SNALP-formulated PLK1424
caused a significant increase in the survival of Hep3B tumor-bearing
mice. This in vivo anti-tumor effect was observed in the absence of any
apparent toxicity or immune stimulation.

[0440] The objectives of this study were as follows: [0441] 1. To
determine the level of mRNA silencing in established Hep3B liver tumors
following a single IV administration of PLK1424 SNALP. [0442] 2. To
confirm the mechanism of mRNA silencing by detecting specific RNA
cleavage products using RACE-PCR. [0443] 3. To confirm induction of tumor
cell apoptosis by histopathology.

[0444] The 1:57 SNALP formulation (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1%
DPPC; and 34.3% cholesterol) was used for this study.

[0446] All samples were filter-sterilized prior to dilution to working
concentration. All tubes were labeled with the formulation date, lipid
composition, and nucleic acid concentration. SNALP samples were provided
at 0.2 mg/ml nucleic acid. A minimum of 2 ml of SNALP was required to
perform the study. Formulations for this study contained:

TABLE-US-00021
[0447] Day 0 Mice will receive Anafen by SC injection (100 μg in 20
μl saline)
immediately prior to surgery. Individual mice are anesthetized by
isoflourane gas inhalation and eye lube applied to prevent excessive
eye drying. While maintained under gas anesthesia from a nose cone,
a single 1.5 cm incision across the midline will be made below the
sternum. The left lateral hepatic lobe is then exteriorized using an
autoclaved cotton wool bud. 25 μl of tumor cells suspended in PBS is
injected into the lobe at a shallow angle using a leur tip Hamilton
syringe (50 μl) and 30G (3/8'') needle. Cells will be injected slowly
(~30 s) and a swab applied to the puncture wound immediately after
needle withdrawal. After any bleeding has stopped (~1 min), the
muscle wall incision is closed with 5-6 sutures. The skin incision is
then closed with 3-4 metal skin clips. Cell suspensions will be
thoroughly mixed immediately prior to each injection. Mice will
recover from anesthesia in a clean cage lined with paper towel and
monitored closely for 2-4 hours. Animals are then returned to normal
housing.
Day 1 All mice will be lightly anesthetized by isoflourane gas and the
sutures examined. Animals will then receive Anafen by SC injection
(100 μg in 20 μl saline).
Day 7 Mice will be randomized into the appropriate treatment groups.
Day 20 Groups A-C: Mice will be weighed and then administered either
PBS, Luc, or PLK1424 SNALP by IV injection via the lateral tail
vein. SNALP will be dosed at 2 mg/kg or equivalent volume (10 ml/kg)
according to body weight.
Day 21 Groups A-C: All mice will be weighed and then euthanized by lethal
anesthesia.
Tumor bearing liver lobes from all mice in each group will be
weighed and collected into RNALater for RNA analysis.
Endpoint: Tumor burden and formulations are expected to be well
tolerated. Mice that exhibit signs of distress associated with the
treatment or tumor burden are terminated at the discretion of the
vivarium staff.
Termination: Mice are anaesthetized with a lethal dose of
ketamine/xylazine
followed by cervical dislocation.
Data Analysis: mRNA analysis of liver tumors by bDNA (QG) assay and RACE-
PCR.
Tumor cell apoptosis by histopathology.

Results

[0448] Body weights were monitored from Day 14 onwards to assess tumor
progression. On Day 20, 6 mice showing greatest weight loss were
randomized into each of the 3 groups and treated. All six mice had
substantial-large I.H. tumors at sacrifice (Day 21). Treatment of the
remaining 14 mice was therefore initiated on the Day 21 (sacrifice Day
22). 10/14 mice had substantial tumors; 2/14 mice had small/probable
tumors; and 2/14 mice had no visible tumor burden.

[0450]FIG. 16 shows that a specific cleavage product of PLK-1 mRNA was
detectable in mice treated with PLK1424 SNALP by 5' RACE-PCR. No specific
PCR product was detectable in mice treated with either PBS or control
(Luc) SNALP. Nucleotide sequencing of the PCR product confirmed the
predicted cleavage site by PLK1424 siRNA-mediated RNA interference in the
PLK-1 mRNA.

[0452] This example illustrates that a single administration of PLK1424
1:57 SNALP to Hep3B tumor-bearing mice induced significant in vivo
silencing of PLK-1 mRNA. This reduction in PLK-1 mRNA was confirmed to be
mediated by RNA interference using 5' RACE-PCR analysis. Importantly,
PLK-1 mRNA silencing by the 1:57 SNALP formulation profoundly disrupted
tumor cell proliferation (mitosis), causing subsequent apoptosis of tumor
cells. As demonstrated in the previous example, this anti-tumor effect
translated into extended survival times in the tumor-bearing mice.

Example 11

Comparison of 1:57 PLK-1 SNALP Containing Either PEG-cDMA or PEG-cDSA in a
Subcutaneous Hep3B Tumor Model

[0453] This example demonstrates the utility of the PEG-lipid PEG-cDSA
(3-N-[(-Methoxypoly(ethylene
glycol)2000)carbamoyl]-1,2-distearyloxypropylamine) in the 1:57
formulation for systemically targeting distal (e.g., subcutaneous)
tumors. In particular, this example compares the tumor targeting ability
of 1:57 PLK-1 SNALPs containing either PEG-cDMA (C14) or PEG-cDSA
(C18). Readouts are tumor growth inhibition and PLK1 mRNA silencing.
The PLK-1 siRNA used was PLK1424 U4/GU, the sequence of which is provided
in Table 8.

[0454] Subcutaneous (S.C.) Hep3B tumors were established in scid/beige
mice. Multidose anti-tumor efficacy of 1:57 PLK-1 SNALP was evaluated for
the following groups (n=5 for each group): (1) "Luc-cDMA"-PEG-cDMA Luc
SNALP; (2) "PLK-cDMA"-PEG-cDMA PLK-1 SNALP; and (3) "PLK-cDSA"-PEG-cDSA
PLK-1 SNALP. Administration of 6×2 mg/kg siRNA was initiated once
tumors reached about 5 mm in diameter (Day 10). Dosing was performed on
Days 10, 12, 14, 17, 19, and 21. Tumors were measured by caliper twice
weekly.

[0455] FIG. 18 shows that multiple doses of 1:57 PLK-1 SNALP containing
PEG-cDSA induced the regression of established Hep3B S.C. tumors. In
particular, 5/5 tumors in the PLK1-cDSA treated mice appeared flat,
measurable only by discoloration at the tumor site.

[0456] FIG. 19 shows the mRNA silencing of 1:57 PLK SNALP in S.C. Hep3B
tumors following a single intravenous SNALP administration. The extent of
silencing observed with the PLK1-cDSA SNALP correlated with the
anti-tumor activity in the multi-dose study shown in FIG. 18.

[0457] The Luc-cDMA SNALP-treated group, which had developed large S.C.
tumors at Day 24, were then administered PLK-cDSA SNALP on Days 24, 26,
28, 31, 33, and 35. There was no additional dosing of the original PLK-1
SNALP-treated groups. The results from this crossover dosing study with
large established tumors is provided in FIG. 20, which shows that
PLK1-cDSA SNALP inhibited the growth of large S.C. Hep3B tumors.

[0458] A comparison of the effect of PEG-cDMA and PEG-cDSA 1:57 SNALPs on
PLK-1 mRNA silencing was performed using established intrahepatic Hep3B
tumors in scid/beige mice. A single 2 mg/kg dose of 1:57 PLK-1 SNALP
containing either PEG-cDMA or PEG-cDSA was administered intravenously.
Liver/tumor samples were collected at 24 and 96 hours after SNALP
treatment. Control=2 mg/kg Luc-cDMA SNALP at 24 hours.

[0459]FIG. 21 shows that PLK-cDMA SNALP and PLK-cDSA SNALP had similar
silencing activities after 24 hours, but that the PLK-cDSA SNALP may
increase the duration of mRNA silencing in intrahepatic tumors.

[0460] FIG. 22 shows the blood clearance profile of 1:57 PLK-1 SNALP
containing either PEG-cDMA or PEG-cDSA. The extended blood circulation
times observed for the PLK-cDSA SNALP may enable the increased
accumulation and activity at distal (e.g., subcutaneous) tumor sites.

[0461] Thus, this study shows that the 1:57 PEG-cDSA SNALP formulation can
be used to preferentially target tumors outside of the liver, whereas the
1:57 PEG-cDMA SNALP can be used to preferentially target the liver.

Example 12

Synthesis of Cholesteryl-2'-Hydroxyethyl Ether

[0462] Step 1: A 250 ml round bottom flask containing cholesterol (5.0 g,
12.9 mmol) and a stir bar was sealed and flushed with nitrogen.
Toluenesulphonyl chloride (5.0 g, 26.2 mmol) was weighed into a separate
100-mL round bottom flask, also sealed and flushed with nitrogen.
Anhydrous pyridine (2×50 ml) was delivered to each flask. The
toluenesulphonyl chloride solution was then transferred, via cannula,
into the 250 ml flask, and the reaction stirred overnight. The pyridine
was removed by rotovap, and methanol (80 ml) added to the residue. This
was then stirred for 1 hour until a homogeneous suspension was obtained.
The suspension was filtered, washed with acetonitrile (50 ml), and dried
under vacuum to yield cholesteryl tosylate as a fluffy white solid (6.0
g, 86%).

[0463] Step 2: Cholesteryl tosylate (2.0 g, 3.7 mmol), 1,4-dioxane (50
mL), and ethylene glycol (4.6 g, 74 mmol) were added to a 100 ml flask
containing a stir bar. The flask was fitted with a condenser, and
refluxed overnight. The dioxane was then removed by rotovap, and the
reaction mixture suspended in water (100 ml). The solution was
transferred to a separating funnel and extracted with chloroform
(3×100 ml). The organic phases were combined, washed with water
(2×150 ml), dried over magnesium sulphate, and the solvent removed.
The crude product was purified by column chromatography (5%
acetone/hexane) to yield the product as a white solid (1.1 g, 69%).

[0464] The structures of the cholesterol derivatives
cholesteryl-2'-hydroxyethyl ether and cholesteryl-4'-hydroxybutyl ether
are as follows:

##STR00009##

[0465] It is to be understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent to
those of skill in the art upon reading the above description. The scope
of the invention should, therefore, be determined not with reference to
the above description, but should instead be determined with reference to
the appended claims, along with the full scope of equivalents to which
such claims are entitled. The disclosures of all articles and references,
including patent applications, patents, PCT publications, and Genbank
Accession Nos., are incorporated herein by reference for all purposes.